Methotrexate (MTX)-resistant sublines were developed by culturing GCH-1 (human gestational choriocarcinoma cell line in vitro) in increasing concentrations of MTX. The parent GCH-1 cells cannot grow at more than 1 X 10(-7)M MTX. The cells which grew at 1 X 10(-7)M MTX and 5 X 10(-7)M MTX are designated GCH-1 : R1 and GCH-1 : RII, respectively. Mechanism of the resistance to MTX has been examined in these lines. The results are as follows: The IC50 of MTX to cells of GCH-1, GCH-1 : RI and GCH-1 : RII was 4.5 X 10(-8)M, 7.5 X 10(-7)M and 9.5 X 10(-7)M, respectively. There was no significant difference between the parent cells and the subline cells in light microscopic findings or secretion of HCG and beta-HCG. A DNA histogram of GCH-1 cells had the main peak at 3n-4n and the second peak at 5n-6n. The second peak diminished after treatment with 5 X 10(-8)M or 1 X 10(-7)M MTX. On the other hand, the DNA histogram of the subline cells had main peak at 3n-4n and second peak at 5n-6n, but the latter was not clear compared with that of the parent cells. The pattern of the DNA histogram of the subline cells didn't change even when they were cultured in MTX free media. Uptake of MTX by the sublines was reduced more than that by the parent line, indicating that affinity of MTX to its carrier component was weakend.(ABSTRACT TRUNCATED AT 250 WORDS)