Site directed mutagenesis has been performed on the gene coding for the beta-subunit of the yeast mitochondrial F1-ATPase. Two different regions were studied. First, the corresponding yeast amino acid, Tyr-344, which was affinity labeled in the bovine enzyme was changed to Phe-344 and Ala-344. The Phe-344 enzyme was completely active and less sensitive to the affinity reagent, 4-chloro-7-nitrobenzofurazan. In contrast, the in vivo level of the Ala-344 enzyme was greatly diminished and apparently inactive. The second region studied is in the glycine rich region homologous in nucleotide binding proteins. Five different replacements were made and all mutations but one completely eliminated the biological activity and reduced the in vivo level of the mutant peptides. These results support the importance of these amino acids in the function of the ATPase.