The sequence requirements for proper transcriptional initiation of the downstream human multidrug resistance MDR1 (P1) promoter were determined using a transient expression system in HeLa cells. The MDR1 promoter has no TATA box and the transcription start site has a strong homology with the initiator (Inr) sequence identified in the murine terminal deoxynucleotidyltransferase (TdT) gene. A deletion analysis showed that sequences from -6 to +11 relative to the P1 transcription start site were sufficient for proper transcriptional initiation, whereas deletion of sequences downstream of +11 resulted in a strong reduction of properly initiated transcripts. In this transient assay system, both the MDR1 and TdT initiator require in Hela cells the presence of an upstream activating sequence such as the SV40 enhancer. This is in contrast to the transcription in in vitro systems, in which the initiator sequence is able to direct transcription in the absence of an enhancer. Analysis of mutations in the initiator sequence from -8 to +10 showed that the A and T nucleotides at position +1 and +3, respectively, were essential, whereas other substitutions in this region had little effect on promoter activity.