To define the cis-acting DNA elements required for rhodopsin expression, we generated lines of transgenic mice carrying sequences upstream of the bovine rhodopsin gene fused to the E. coli β-galactosidase gene (IacZ). Upstream sequences extending from -2174 to +70 bp, from -734 to +70 bp, and from -222 to +70 by direct photoreceptor-specific expression. All three -2174 lines demonstrate a superior-temporal to inferior-nasal gradient of expression across the retina, whereas lines carrying the shorter constructs demonstrate either spatially continuous expression across the retina, discrete clusters of expression, or both. As a complementary approach to defining regulatory elements, we compared DNA sequences 5′ of the murine, bovine, and human rhodopsin genes. Significant homology between all three species was found just upstream of the transcription start site and at approximately 1.5 kb upstream. © 1991.