We have isolated a 4.0-kb fragment from a genomic library of Candida albicans which contained two open reading frames (ORFs). One of them is homologous to a prolyl-tRNA synthetase that catalyses the charging of a specific tRNA by proline (CaPRS). A deduced sequence of 575 amino acids representing a polypeptide of 66.2 kDa was determined. A FASTA search indicated that the CaPRSp had an overall similarity of 54.4% with the product of a Saccharomyces cerevisiae ORF (YER087) and 43.8% with the prolyl-tRNA synthetase of Escherichia coli (COLIPRO). Consensus Class II aminoacyl-tRNA synthetase sequences were identified by the PROSITE program. CaPRS was localized to chromosome R of the C. albicans genome and CaPRS DNA hybridized to a major RNA transcript of 1.7 kb under all conditions tested.