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Early studies by Howes et al. (1) indicated that after wounding of skin there is a "lag phase" of about four to six days before significant fibroplasia and increased wound tensile strength could be detected. The existence of a "lag phase" was supported by Ross and Benditt's observation that there was no histological evidence of collagen fiber formation in open guinea pig wounds until the third day after wounding (2) and Madden and Peacock's observation that hydroxyproline synthesis did not increase until the fourth day in sutured rat wounds (3). In contrast, our studies have suggested that actual collagen synthesis in open rat skin wounds increases as early as 24 hr after wounding (4, 5). These observations raised the question as to what were the actual rates and kinetics of collagen synthesis early after wounding. In addition, because Ross noted that the peak number of collagen-syn t hesizing wound fibroblasts was not obtained in the wound until day 6 (2, 6), we sought to identify which wound area synthesized collagen as early as 24 hr after wounding. In the present study, collagen synthesis was found to be significantly increased in open rat skin wounds by 24 hr and the activity was localized in the panniculus carnosis.Methods. Wounding procedure: Six to ten open wounds were made on shaved backs of male Sprague-Dawley rats (1 50-180 g) with either a 3 or 6 mm dermal punch through the level of the panniculus carnosis. The punched-out skin was discarded. On various days after wounding, the animals were sacrificed by decapitation and the entire dorsal skin, including the panniculus carnosis, was removed. Either a 4 or 8 mm dermal punch was then used to excise the entire wound and normal skin samples from the animal pelt.Collagen synthesis. The excised wounds or Supported by NIH Grant No. GM-20298. normal skin samples were minced and incubated in 3 ml of Krebs-Ringer medium containing 10 pCi 3H-5 proline (42 Ci/mM, Schwarz/Mann, Orangeburg, NY) as we have described previously (4). Duplicate analyses were made on all animals. Following a 2-hr incubation at 37" under normal atmosphere, the samples were removed, frozen, homogenized and radioactive protein was precipitated at 4" with trichloroacetic acid (5%). The radioactive protein was then digested by purified bacterial collagenase to separate collagen from noncollagen protein (4). The radioactivity released by bacterial collagenase as well as the undigested noncollagen protein were each measured by liquid scintillation spectrometry. The absolute amount of collagen synthesized during this 2hr period is reported as radioactivity per mg dry tissue weight. In addition, the relative amount of collagen synthesized was calculated by comparing absolute collagen synthesis to noncollagen protein synthesis (bacterial collagenase-resistant protein). A calculation was used to correct for the enriched imino acid content of collagen compared to other proteins (4). The student's t test was used for statistical analyses.Groups for study of collagen synthesis in open rat wounds. ...