Carotid body type I cells and the O2 sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2 tension causes inhibition of an outward rectifying the O2-sensitive potassium (K) channel in the O2-sensitive pheochromocytoma (PC12) cell line, which leads to membrane depolarization and increased intracellular free Ca2+. We found that removal of Ca2+ from the extracellular milieu, inhibition of voltage-dependent Ca2+ channels, and chelation of intracellular Ca2+ prevents full activation of the TH gene expression during hypoxia. These findings suggest that membrane depolarization and regulation of intracellular free Ca2+ are critical signal transduction events that regulate expression of the TH gene in PC12 cells during hypoxia. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O2 tension in both type I cells and PC12 cells. The increase in TH gene expression in PC12 cells during hypoxia is due to increases in both the rate of transcription and mRNA stability. Analysis of reporter-gene constructs revealed that increased transcription of the TH gene during hypoxia is regulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of the gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays revealed that hypoxia-induced binding occurs at both the AP1 and HIF-1 sites. Results from super-shift and shift Western assays showed that a heterodimer consisting of c-Fos and JunB binds to the AP1 site during hypoxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also found that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O2 tension.