Neurespora c issa ha been utlzed extensively in the study of (5,9,13), and the genetic analysis of mutations affecting period lengths (6-8). In addition, circadian rhythms have been found in a variety of biochemical parameters, including pyrdine nucleotides (1), 5'-AMP levels (2), fatty acids (12), RNA and DNA synthesis (11), and several enzymes (10).The present study arose from the need to define the Neurospora system more fully and to develop new techniques for further analysis of the clock. Difficulties in obtaining sufficient culture 'This work was supported by National Institute of General Medical Sciences Grant GM-22144.material for the assay of certain biochemical parameters, the need to develop a reliable method of pulsing drugs, and the need to know more about the physiology of the Neurospora clock system were among the factors considered in developing this investigation. Specifically, we wished to answer the questions of whether it was possible to determine the phase of the clock in a small portion of the mycelium rather than in the entire culture, and additionally, whether there is a functional clock in hyphae behind the growing front.The latter question is significant in light ofreports ofNeurospora aging (20, 21), which have indicated that the growing tips of a culture are metabolically active, contain large numbers of nuclei and active mitochondria, and show considerable cytoplasmic streaming, while, even a short distnce behind this growing front (1-2 cm), the hyphae are metabolically less active, filled with fat globules and vacuoles, contain few nuclei or mitochondria, and show little cytoplasmic streaming. Because the growing front must differentiate to form aerial hyphae and conidia at the appropriate time in the circadian cycle, it is evident that temporal information is available in this region. The region behind the growing front remains essentially unchanged, undergoing no significant differentiation during subsequent circadian cycles. Therefore, the existence or nature of temporal information in this older portion of the culture cannot be directly determined simply by observing the culture.Preliminary studies in this laboratory indicated that small portions of mycelium cut from the growing front and transferred to fresh medium showed a circadian rhythm with a phase essentially the same as that shown by the growing front of the intact original culture (3). We report here a method for assaying the phase of mycelium in any region of the culture and a detailed analysis of the results of such assays. MATERIALS AND METHODSStains. The band,csp-l strain of Neurospora crassa (obtained from S. Brody) was used in all experiments. The csp-l mutation prevents the separation of conidia from the hyphae and thus prevents spreading of conidia during transfer operations (18).Medium. Acetate-casamino acids medium (Vogel's [19] salts, 1.2% sodium acetate, 0.05% casamino acids, 2% agar) was added to race tubes 220 mm long and 14 mm in diameter (8.3 ml/tube) and to Petri dishes 150 mm in diameter a...