cGMP-phosphodiesterase (PDE) is the key effector in rod photoreceptor signal transduction. Mutations in the gene encoding its catalytic β-subunit (β-PDE) cause retinal degenerations leading to blindness. We report that the short −93 to +53 sequence in the upstream region of this gene is sufficient for β-PDE transcription in both Y79 human retinoblastoma cells and Xenopus embryo heads maintainedex vivo. This sequence also functions as a minimal rod-specific promoter in transgenic Xenopustadpoles. The Nrl transcription factor binds in vitro to the βAp1/NRE regulatory element located within this region and transactivates it when overexpressed in nonretinal 293 embryonic kidney cells. We also found a G/C-rich activator element, β/GC, important for promoter activity in Y79 retinoblastoma cells andXenopus embryos. Both the ubiquitous Sp1 and the central nervous system-specific Sp4 transcription factors are expressed in retina and interact with this element in vitro. Electrophoretic mobilities of β/GC-Y79 nuclear protein complexes are altered by antibodies against Sp1 and Sp4. Thus, our results implicate Nrl, Sp1, and Sp4 in transcriptional regulation of the rod-specific minimal β-PDE promoter. We also conclude thatXenopus laevis is an efficient system for analyzing the human β-PDE promoter and may be used to study other human retinal genes ex vivo and in vivo.