Previous transgenic mouse studies demonstrated that the bovine rhodopsin sequence between -222 and +70 base pairs (bp) contains a minimal promoter, which is sufficient to direct photoreceptor cell-specific expression of a lacZ reporter gene. To more fully define the DNA regulatory elements and protein factors involved in regulating rhodopsin transcription, we have developed an in vitro transcription system derived from bovine retinal nuclear extracts. Retinal extracts, as compared to liver, HeLa, and Drosophila embryonic cell extracts, demonstrated preferential activity for the rhodopsin promoter. A template spanning the bovine rhodopsin upstream region from -590 to +15 bp showed significant activation relative to the basal activity seen with a TATA box containing -38 to +15 bp template. Deletion analysis indicated that the region between -85 and -38 bp contained significant positive regulatory activity. This activity was not observed with HeLa extracts, suggesting that it might be retina-specific. Systematic site-directed mutagenesis of the subregion from -64 to -38 bp indicated that sequences between -60 and -58 bp and between -48 and -40 bp harbor critical elements. The former sequence is part of the binding site for the retina-specific transcription factor Nrl, which has been implicated in rhodopsin regulation. Electrophoretic mobility shift assays showed that the latter sequence (-48 to -40 bp), and flanking DNA, designated Ret 4, is bound by both retina-specific and ubiquitously expressed protein factors. Shift assays with mutant oligomers further defined the putative recognition sequences for these protein factors. Together, our results suggest that multiple promoter elements and transcriptional factors are involved in regulating photoreceptor-specific rhodopsin transcription.