Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells. We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this. In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported. Two genes for L41 protein, named L41a and L41b, independently present in the genome of S. cerevisiae, were isolated. L41-related genes were also isolated from a few other yeast species. Each of these genes has an intron at the same site of the open reading frame. Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S. cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline. This was confirmed by constructing a cycloheximide-resistant strain of S. cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon.