The stimulatory effects of glucose and lactate on protein synthesis by round spermatids (steps 1-8) were further studied. When the cells were incubated with lactate, the response of protein synthesis in round spermatids was closely related to the intracellular level of ATP. The ATP level in spermatids increased to 3.13 +/- 0.20 nmol/10(6) cells from 0.37 +/- 0.02 nmol/10(6) cells after incubation of the cells at 34 degrees C for 60 min in the presence of lactate (20 mM). However, the ATP level fell rapidly to an undetectable level (less than 0.02 nmol/10(6) cells) during incubation for 30 min at 34 degrees C without lactate. The ATP level and the rate of protein synthesis in spermatids increased rapidly when lactate (20 mM) was added to the control cells during incubation. It was also found that incorporation of 32P into ATP was increased by treatment with lactate (20 mM), but glucose (10 mM) had no effect on 32P incorporation into ATP. When the rates of utilization of glucose and lactate by spermatids were examined, using 14C-labeled glucose and lactate, the rate of utilization of lactate was faster than that of glucose. These results suggest that the ATP is probably a major factor in the stimulation of protein synthesis in round spermatids.