GTP cyclohydrolase I (GCH, EC 3.5.4.16) regulates the level of tetrahydrobiopterin and in turn the activities of nitric oxide synthase and aromatic amino acid hydroxylases. Type II GCH mRNA, an alternatively spliced species abundant in blood cells, encodes a truncated and nonfunctional protein. When we stimulate peripheral blood mononuclear cells by PHA, the transcription of full-length GCH mRNA increased, but that of type II mRNA decreased transiently. We further demonstrated that the type II cDNA exerted a dominant-negative effect on the wild-type cDNA, similar to the effect of some GCH mutants. Therefore, type II mRNA may regulate GCH and then contribute to the regulation of NO production by BH4-dependent iNOS in mononuclear cells. Selection of the splicing sites may be coupled with transcriptional activation of the GCH gene.