The potential of a combination of differentiation induction and chemotherapy was analyzed. Treatment of the murine embryonal carcinoma (EC) cell line PCC4in vitro with all-transretinoic acid (RA) was followed by exposure to cisplatin (CDDP) or etoposide (VP-16). The expression of EC-cell-specific markers decreased upon 96 hr exposure to 10−9, 10−8, 10−7and 10−6M RA, indicating a loss of embryonal phenotype of the cells, whereas expression of markers specific for cytokeratins and neurofilaments was increased after this treatment. These data suggest early somatic differentiation of PCC4 cells upon treatment with RA. Cellular growth rate of PCC4 cells was not affected by preincubation with RA at 10−9 M for 96 hr, but was reduced at 10−8 and 10−7 M RA and inhibited at 10−6 M RA. Culture of PCC4 cells in the presence of 10−7 M RA for 96 hr led to accumulation in G1 of the cell cycle, whereas at 10−8 M RA cell-cycle distribution was not affected. RA-treated and -untreated PCC4 cells were compared for CDDP and VP-16 sensitivity. Pre-treatment with 10−9, 10−8 and 10−7 M RA increased CDDP sensitivity, resulting in a 1.9-, 2.7- and 2.6-fold decrease in the concentrations inhibiting survival by 50% (IC50s) respectively. Pre-treatment with 10−8 and 10−7 M RA increased VP-16 sensitivity 2.5- and 3.0-fold, respectively. Enhanced CDDP sensitivity at RA concentrations not affecting cell-cycle distribution was not attributable to changes in cellular platinum (Pt) accumulation, or to changes of Pt-DNA binding. Enhanced VP-16 sensitivity also occurred while topoisomerase-II activity was unchanged and topoisomerase-I activity was increased 2-fold. Furthermore, there was a 2.0- to 4.5-fold increase in the cellular concentration of VP-16 after 10−8 and 10−7 M RA pre-treatment. In conclusion, this study shows that in PCC4 cells the rationale for combining RA and VP-16 or CDDP is 2-fold: RA induces differentiation and increases CDDP and VP-16 sensitivity.