Abstract Male rats (30–35 days old) were utilized to examine the process of depletion and replenishment of the testicular cytosolic estrogen receptor and to investigate the effects of cycloheximide and actinomycin D on these processes. The dose dependence and temporal nature of receptor depletion and replenishment were investigated. Maximum depletion (> 90%) occurred by 1 h after in vivo administration of either 5 or 10 μg estradiol-17β. Depletion of the cytosolic receptor at 1 h occurred concomitant to a marked increase in nuclear receptor thus indicating translocation. Receptor replenishment to control levels was observed by 6 h post treatment. To determine the requirements for transcriptional and translational events in the replenishment process, actinomycin D and cycloheximide were administered in vivo. Simultaneous treatment with cycloheximide and estradiol resulted in a significant inhibition of replenishment at 6 and 12 h post treatment of 46 and 60% below control levels, respectively. Cycloheximide treatment alone had no effect on receptor levels. A significant inhibition of replenishment at 6 h was also shown when cycloheximide was given 3 h after estradiol treatment. Cycloheximide administration at 6 h after estradiol significantly suppressed receptor levels at 12 h suggesting that replenished receptor levels at 6–12 h are in a state of rapid turnover. Receptor replenishment exhibited a different response to actinomycin D treatment in that significant inhibition was observed only when the drug was administered at 3 h after estradiol treatment. These results demonstrate that testicular cytosolic estrogen receptor depletion is dose and time dependent. The results further demonstrate that receptor replenishment involves protein synthesis and suggests that synthesis of new RNA might also be required.