The overproduction of P-glycoprotein (Pgp) has been associated with the development and maintenance of the multidrug resistant (MDR) phenotype, although the regulatory events responsible have not yet been elucidated. We have analyzed the overexpression of the TATA-less hamster class-I Pgp-encoding gene (Pgp1) in several MDR Chinese hamster cell lines. The MDR lung cell line DC-3F/VCRd5L, as well as the MDR ovary cell line CHRC5, express a level of Pgp1 RNA commensurate with the increase in Pgp1 dosage; in contrast, the actinomycin D (ActD)-selected sublines of DC-3F overexpress Pgp1 mRNA without a concomitant increase in Pgp1 gene-copy number. Analysis of Pgp1 transcription start point (tsp) utilization revealed that drug-sensitive DC-3F cells, as well as DC-3F/VCRd5L and CHRC5 cells, utilize one major tsp; in contrast, the ActD-resistant sublines 'switch' to a more complex pattern, using four additional Pgp1 tsp 32, 42, 52, and 67 bp downstream from the major parental tsp (+1). This observation of a difference in the regulation of transcription of Pgp in MDR vs. drug-sensitive cells suggests that the 'switch' in tsp selection may be involved in the increased expression of Pgp1 mRNA. Interestingly, despite the existence of several hundred MDR cell lines, very few have been analyzed with respect to tsp selection; it is therefore possible that alternate tsp selection is a relatively common yet heretofore unobserved component of the MDR phenotype. Moreover, these cells provide an excellent system in which to evaluate the sequence elements and protein factors that govern the selection of tsp in TATA-less promoters.