Cassette site-directed mutagenesis was employed to generate mutations in the a subunit (uncB (a) gene) of F1F0ATP synthase. Using sequence homology with similar subunits of other F1F0ATP synthases as a guide, 20 mutations were targeted to a region of the a subunit thought to constitute part of the proton translocation mechanism. ATP-driven proton pumping activity is lost with the substitution of lys, ile, val, or glu for arginine 210. Substitution of val, leu, gln, or glu for asparagine 214 does not completely block proton conduction, however, replacement of asparagine 214 with histidine does reduce enzyme activity below that necessary for significant function. Two or three mutations were constructed in each of four nonpolar amino acids, leucine 207, leucine 211, alanine 217, and glycine 218. Certain specific mutations in these positions result in partial loss of F1F0ATP synthase activity, but only the substitution of arginine for alanine 217 reduces ATP-driven proton pumping activity to undetectable levels. It is concluded that of the six amino acids studied, only arginine 210 is an essential component of the proton translocation mechanism. Fractionation of cell-free extracts of a subunit mutation strains generally reveals normal amounts of F1 specifically bound to the particulate fraction. One possible exception is the arginine 210 to isoleucine mutation which results in somewhat elevated levels of free F1 detectable in the soluble fraction. For nearly all a subunit mutations, F1F0-mediated ATP hydrolysis activity remains sensitive to inhibition by dicyclohexylcarbodiimide in spite of the fact that the mutations block proton translocation.