NAD kinase and NADP phosphatase activities were detected in the supernatant and the pellet fractions prepared by sonication and centrifugation of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis. A detailed study of substrate concentration-velocity curves enabled us to define the saturating substrate concentrations that were used in the enzyme assays. An analysis of the reproducibility of the entire assay procedure indicated that the pooled standard error was about 14%. We report circadian variations in the activities of NAD kinase and NADP phosphatase in the soluble and membrane-bound fractions of both synchronously dividing and nondividing cultures maintained in constant darkness. Bimodal circadian rhythms in total NADP phosphatase activity were found in dividing cells (peaks at circadian times [CT] 00 and 12). The peak observed at CT 00-03 disappeared when the cells had ceased dividing, a result that suggests that it might be regulated by the cell division cycle. NAD kinase activity displayed unimodal circadian rhythms (peak at CT 12) in dividing cells, which persisted with the same phase after the culture entered the stationary phase of growth. Results are discussed with reference to a model (K. Goto, D. L. Laval-Martin, and L. N. Edmunds, Jr., 1985, Science 228, 1284-1288) in which we have proposed that the Ca2(+)-transport system, Ca2+, calmodulin, NAD kinase, and NADP phosphatase could represent clock "gears" that might constitute a self-sustained circadian oscillating loop.