The active site of galactose-1-phosphate uridylyltransferase (GALT) includes a HPH sequence that has been conserved in all species examined from Escherichia coli to humans. The crystal structure of the E. coli enzyme suggests that this proline is important in positioning the active site histidine (His-166) near the substrate. To examine the role of this proline in the homologous human sequence, we have performed saturating mutagenesis at Pro-185 within human GALT and characterized each resultant mutant enzyme using a yeast expression system. Activity analyses in crude lysates indicated that only proline at position 185 produced wild-type levels of activity, although five other amino acids, Ala, Gly, Ser, Gln, and Glu, all produced partially active enzymes. Western blot analyses of the GALT proteins in these lysates demonstrated that abundance varied from 9-118% of wild-type and was independent of activity. All five active mutant proteins were purified and characterized with regard to specific activity, apparent Km for both substrates, and temperature-dependence of activity. Finally, modeling of these mutations onto the conserved E. coli active site structure was performed. Together, these results provide functional evidence demonstrating the critical role of Pro-185 in facilitating the transferase reaction.