We carried out cytogenetic studies of four Chinese hamster, mouse, and human cell lines selected for high levels of resistance (500- to 4,000-fold) to vincristine (VCR) by a multistep selection procedure. All cells examined contained gene amplification-associated metaphase chromosome abnormalities, either homogeneously staining regions (HSRs), abnormally banding regions (ABRs), or double-minute chromosomes (DMs); control actinomycin D- and daunorubicin-resistant hamster lines did not exhibit this type of chromosomal abnormality. VCR-resistant Chinese hamster sublines exhibited both increased synthesis of the protein V19 (Mr 19,000; pl = 5.7) and increased concentrations of V19 polysomal mRNA. When VCR-resistant cells were grown in drug-free medium, level of resistance, synthesis of V19, and amount of V19 mRNA declined in parallel with mean length of the HSR or mean number of DMs per cell. Cross-resistance studies indicate that VCR-resistant cells have increased resistance both to antimitotic agents and to a wide variety of agents unrelated to VCR in chemical structure and/or mechanism of action. Our studies of tubulin synthesis in Chinese hamster cells indicate no overproduction of tubulin or presence of a mutant tubulin species. Comparison with antifolate-resistant Chinese hamster cells known to contain amplified dihydrofolate reductase genes localized to HSRs or ABRs strongly suggests that the HSRs, ABRs, or DMs of the Vinca alkaloid-resistant sublines likewise represent cytological manifestations of specifically amplified genes, possibly encoding V19, involved in development of resistance to VCR.