We have studied retina-specific gene expression and gene promoter activity in WERI-Rb1 retinoblastoma cells. In general, the expression of endogenous genes matched the efficiency of promoter activity of the transfected gene: interphotoreceptor retinoid binding protein and phosphodiesterase-beta mRNAs and reporter activities were readily detected while other retina-specific messages were at or below the detection limit in WERI-Rb1 cells. Phosphodiesterase-beta promoter appeared active in all six cell lines tested. The viral SV40 promoter is very weak in WERI-Rb1 cells, which has implications for its use in gene constructs targeted to the photoreceptors. Our results also show that polyethyleneimine 25 is an efficient and simple carrier for DNA. The optimized transfection conditions permit the use of 24-well plates and low amounts of DNA for improved analysis of promoter activities, as compared to previous studies. Our results are expected to facilitate further research on retina-specific gene expression.