The characterization (Ritter, J.K., Chen, F., Sheen, Y. Y., Tran, H.M., Kimura, S., Yeatman, M.T., and Owens, I. S. (1992) J. Biol. Chem. 267, 3257-3261) of the single-copy UGT1 gene complex locus encoding both bilirubin and phenol UDP-glucuronosyltransferases (transferase) has been critical to the determination of genetic defects in Crigler-Najjar patients. The complex (UGT1A-UGT1M) codes for at least two bilirubin, three bilirubin-like, and eight phenol transferase isozymes. In the 5' region, a minimum of 13 different exons 1, each with an upstream promoter, are arrayed in series with 4 common exons in the 3' region of the locus. Each exon 1 encodes the amino terminus of a transferase, and the common exons encode the common carboxyl terminus of each isoform. Although a deleterious mutation in a common exon inactivates the entire locus, a deleterious mutation in an exon 1, as we report here for the UGT1A gene in a Crigler-Najjar Type I patient, affects the amino terminus of that single isoform. Recessively inherited mutant alleles for the predominant bilirubin isozyme, the HUG-Br1 protein, substituted Arg for Gly at codon 276 (G276R) in exon 1 of UGT1A abolishing a conserved di-glycine. The mutant HUG-Br1-G276R protein expressed in COS-1 cells had no detectable bilirubin glucuronidating activity at either pH 7.6 or 6.4. Although each of the bilirubin-type isozymes contains a conserved peptide between residues 270 and 288, all UDP-glucuronosyltransferases contain a di-glycine at approximately position 276-277, making it strictly conserved. Structure-function relationship was studied by site-directed mutations of the HUG-Br1 cDNA; G276A, G276Q, G276E, G276I, and P270G mutants were inactive, and V2751- and P285G-altered transferases expressed normal activity. Conservation of residues between the related baculoviral ecdysone UDP-glucosyltransferase and the UDP-glucuronosyltransferases confirms the critical role of the Gly-276 as well as other residues.