Punch (Pu), the gene encoding the pterin biosynthetic enzyme GTP cyclohydrolase in Drosophila, is a complex locus. Mutations fall into several complementation classes that correspond to classes of mutants with distinct morphological and protein phenotypes. Two of these classes are developmentally specific, with mutants in each having defects in discrete subsets of the known functions of the locus. Defined functions of the locus include a role in embryonic nuclear divisions using initially a maternal Pu product, the synthesis of pterin cofactors that are required for catecholamine biosynthesis beginning in late embryogenesis, and the production of pterin-screening pigments in the developing adult eye. Mutant phenotypes include an interruption in synchronous nuclear divisions in precellular blastoderm embryos, a segment pattern phenotype in late embryos, failure to pigment and cross-link embryonic cuticular structures and failure to synthesize red eye pigments. Molecular analysis reveals that the locus is large, a minimum of 29 kb as defined by Southern mapping of Pu mutants. This region is transcriptionally extremely active, encoding at least 16 developmentally regulated transcripts. One transcript has been shown to be responsible for the production of the adult eye GTP cyclohydrolase on the basis of developmental profile, location with respect to the mapping of eye-specific Pu mutants, absence in eye-specific mutants, and hybrid-selection in vitro translation experiments. Several other transcripts are candidates for Pu vital functions, as suggested by their pattern of expression and their derivation from regions to which lethal Pu mutations map.