Exposure of MOLT-3 human leukemic cells in culture to a lipophilic antifolate, trimetrexate (TMQ), resulted in the development of sublines resistant to antifolates as well as to drugs related to multidrug resistance. The TMQ-resistant sublines had an increase in dihydrofolate reductase (DHFR) activity and overexpression of P-glycoprotein. In these sublines, neither the DHFR gene nor the MDR1 gene were amplified. In these cells, DHFR transcripts were also not overexpressed but DHFR protein was increased, indicative of translational or post-translational control of DHFR activity. In contrast, MDR1 transcripts were found to be overexpressed, in parallel with P-glycoprotein production. Therefore, increases in P-glycoprotein appear controlled at the transcriptional level. These data support evidence that TMQ produced two phenotypic changes independently: the former probably from folate deficiency and the latter from the lipophilic nature of the compound.