The human D1A dopamine receptor is transcribed from a tissue-specific regulated gene under the control of two promoters. An activator region (AR1) located between nucleotides -1154 and -1136 (relative to the first ATG) enhances transcription from the upstream promoter that is active in the brain. In this investigation, we sought to identify the nuclear factors that regulate the D1A gene through their binding to AR1 using yeast one-hybrid screening. Sp3 and Zic2 were among the positive clones isolated. Although Sp1 was not isolated from this screening and purified Sp1 alone does not bind to AR1 in gel shift experiments, this general transcription factor binds to AR1 in the presence of D1A expressing NS20Y nuclear extract and activates the D1A promoter. Thus, Sp1 appears to require an unknown factor(s) or post-translational modification to interact with AR1. On the other hand, Zic2 and Sp3 inhibit Sp1-induced activation of the D1A gene in an AR1-dependent manner. Zic2 and D1A genes have reciprocal brain regional distributions; Zic2 is expressed primarily in the cerebellum, and D1A is highly expressed in corpus striatum. These observations collectively suggest that one of the physiologic functions of Zic2 is repression of D1A gene transcription and that the intracellular balance among Sp1, Sp3 and Zic2 is important for regulating the tissue-specific expression of this dopamine receptor.