The Saccharomyces cerevisiae Gas1 protein is synthesized as a precursor with a hydrophobic extension at the carboxyl terminus which is removed and replaced with an inositol containing glycolipid that anchors the protein to the plasma membrane. We performed saturation mutagenesis on the anchor attachment site (Asn506) and showed that only a subset of amino acids with small side chains could act as substrates for peptide cleavage and glycolipid addition. After Asn, which is the most efficient anchor attachment site, Ser, Gly, Ala, Asp, and Cys function with decreasing effectiveness. Mutational analysis also revealed that the 2 adjacent amino acids to the carboxyl side of the anchor attachment site are important for efficient anchoring. These two amino acids should have relatively short side chains with the second position being more critical. Analysis of the region between the anchor attachment site and the carboxyl-terminal hydrophobic region indicated that this region may not simply perform a spacer function.