Human choriocarcinoma cells in vitro (ENAMI-1) were exposed to various concentrations of MTX (10(-8)M, 10(-7)M, 10(-6)M, 10(-4)M) for 48 hr, and after removal of MTX, cells were harvested and measured by FCM every 24 hr for 96 hr. Although the growth of cells were inhibited in MTX more than 10(-7)M in concentration, hCG-beta levels were elevated markedly. It was showed that hCG positive cells were accumulated in G1-phase by flow cytometric two color analysis with monoclonal antibody. In the presence of MTX more than 10(-7)M in concentration, the cells were accumulated in S-phase. After removal of 10(-7)M MTX, S-phase cells migrated toward G2 + M phase on the other hand in the presence of MTX more than 10(-6) M in concentration cells stopped moving to G2 + M phase. Immunoperoxidase with monoclonal antibodies against BrdU and hCG-beta has got the same phenomena resulted by FCM. It was confirmed that hCG was an useful marker for choriocarcinoma.