To understand the mechanisms that control the cell-specific visual pigment gene transcription, the Xenopus rhodopsin 5′ regulatory region has been characterized in vivo using transient transfection of Xenopus embryos and transgenesis. The principal control sequences were located within -233/+41, a region with significant conservation with mammalian rhodopsin genes. DNase footprinting indicated seven distinct regions that contain potential cis-acting elements. Sequences near the initiation site (-45/+41, basal region) were essential, but not sufficient, for rod-specific transcription. Two negative regulatory regions were found, one between -233 to -202, with no apparent similarity to known elements, and a second Ret-1-like CAAT (-136/-122) motif. Deletion of either sequence led to a 2-3-fold increase in expression levels, without a change in rod specificity. Sequences between -170 to -146, which contain an E-box motif, were necessary for high level expression in transgenic tadpoles but not in transient transfections. Sequences between -84 and -58, which contained an NRE-like consensus were found to be necessary for high level expression in both assays. Although expression levels were modulated by various proximal sequences in the rhodopsin promoter, none of the tested sequences were found to be necessary for rod specificity. Promoter constructs with a consensus BAT-1 sequence in conjunction with an NRE-like element upstream of the basal promoter directed low level green fluorescent protein expression in the central nervous system in transgenic tadpoles. These results suggest that rod cell-specific expression of rhodopsin is controlled by redundant elements in the proximal promoter.