Various factors that influence in vitro pairing of Echinostoma revolutum adults were studied. More worms paired in Locke's, Ringer's, and 0.85% NaCl solutions than in the defined medium NCTC 135. Also, more pairing occurred at 39 or 43 degrees C than at 35 degrees C. Echinostomes placed 1, 3, or 5 cm apart in 5.5 cm diameter Petri dishes as well as those placed 2, 4, or 6 cm apart in 8.5 cm diameter Petri dishes paired; however, worms placed 8 cm apart in the latter dishes did not. Individuals maintained in Locke's solution at 20 or 39 degrees C for 1 h after having been removed from the chicks paired less frequently than those used for experiments immediately after their removal from chicks. Worms showed more pairing in fresh Locke's solution than in this solution preconditioned by excretory-secretory products of E. revolutum. Scanning electron microscopy revealed no differences between the tegument of fresh worms and of those maintained under experimental conditions for 2 h at 39 degrees C.