The effects of serial and single administration with actinomycin D (ACT-D) and chromomycin A3 (CHRM) in mice submandibular gland (SMG) were examined by quantitative analysis and immunostaining for epidermal growth factor (EGF), morphometory, andautoradiography. Testosterone (TP) administration was carried out in the same groups of mice and compared with the groups administrated ACT-D and CHRM alone.In the normal SMG of mice immunohistochemically detectable EGF was localized in granular convoluted tubule (GCT) cells. EGF level in male SMG was 10 to 25 times greater than in female or castrated mice. By the serial administration of ACT-D and CHRM, EGF staining was still confined to GCT cells but cell-to-cell variations of EGF staining became move conspicuous, and the ratio of “EGF positive cells”/SMG, and EGF level in SMG were significantly decreased. In serial TP administrated group, GCT cells increased in both size and numbers, and EGF levels in their SMGs were much higher than in the normal and strong EGF staining was found in GCT cells. In the groups that ACT-D or CHRM were used together with TP, the ratio of GCT/SMG and “EGF positive cells”/SMG, and EGF concentration in SMG were almost same as in the normal.In the normal SMG of female mice, a small number of nuclei in both intercalated and acinar cells were labeled with 3H-thymidine. Incorporation of 3H-uridine was little higher in acinar cells than in GCT cells. By the single injection of TP, incorporations of both precursors were increased and the peaks of incorporation of 3H-thymidine and 3H-uridine were the second and the third day after injection, respectively. At that time, 3H-thymidine labeled nuclei existed in some GCT cells and 3H-uridine was found in both GCT and acinar cells in almost the same frequency. When ACT-D or CHRM were used together with TP, incorporations of precursors were almost the same as in the normal.The present results suggest that ACT-D and CHRM inhibit not only RNA synthesis but also EGF and DNA synthesis in mice SMG. Furthermore, ACT-D and CHRM may disturb the effects of TP in GCT cells.