Carboxypeptidase G2 (CPG2), an enzyme produced by Pseudomonas strain RS-16, degrades folates as well as methotrexate and other folic acid analogs such as aminopterin and dichloromethotrexate, but not the "non-classical" folate antagonist trimetrexate (TMTX). The possibility of enhancing TMTX cytotoxicity of CPG2 induced depletion of intracellular folates was investigated in human leukemic CCRF-CEM cells and in three methotrexate resistant sublines of these cells with different mechanisms of resistance, CCRF-CEM/E, a subline with increased DHFR; CCRF-CEM/P, a subline defective in polyglutamylation and CCRF-CEM/T, a subline with impaired MTX uptake. The cytotoxic effect was detected using a colorimetric assay with MTT. Dose-effect relationships of single drugs alone and in combination were analyzed by the median effect principle and by the combination indices for the quantitation of synergism or antagonism with the aid of a microcomputer. TMTX alone was very effective on the parent and all the resistant cell lines (CCRF-CEM/E, CCRF-CEM/P, CCRF-CEM/T) with ED50 values in the nanomolar range (1.4, 1.6, 1.5 and 0.7 nM, respectively) following 5 days of exposure. The ED50s of CPG2 for these cell lines were 3.5, 2.6, 26.6 and 7.9 X 10(-5) U/ml, respectively. A synergistic cytotoxic effect of TMTX after simultaneous continuous exposure was observed with CPG2 on CCRF-CEM cells and on the three resistant cell lines.