Genes encoding the G alpha subunit were cloned from Mucor circinelloides, a zygomycete dimorphic fungus. There are at least four genes that encode for G alpha subunits, gpa1, gpa2, gpa3, and gpa4. The genes gpa1 and gpa3 were isolated and characterized, and their predicted products showed 36%-67% identity with G alpha subunits from diverse fungi. Northern blot analysis of gpa3 showed that it is present in spores and constitutively expressed during mycelium development and during yeast-mycelium and mycelium-yeast transitions. However, during yeast cell growth, decreased levels of mRNA were observed. Sequence analysis of gpa3 cDNA revealed that Gpa3 encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40.8 kDa. The deduced sequence of Gpa3 protein contains all the consensus regions of G alpha subunits of the G alpha(i/o/t) subfamily except the cysteine near the C terminus for potential ADP-ribosylation by pertussis toxin. This cDNA was expressed in Escherichia coli and purified by affinity chromatography. Based on its electrophoretic mobility in SDS-PAGE, the molecular mass of the His(6)-tagged Gpa3 was 45 kDa. The recombinant protein was recognized by a polyclonal antibody against a fragment of a human G alpha(i/o/t). Furthermore, the recombinant Gpa3 was ADP-ribosylated by activated cholera toxin and [P-32]NAD but not by pertussis toxin. These results indicate that in M. circinelloides the G alpha subunit Gpa3 is expressed constitutively during differentiation.