Five putative phosphoinositide-specific phospholipases C (PLC) genes were identified in three species of filamentous fungi. Using polymerase chain reaction with degenerate oligonucleotide primers, gene fragments encoding amino acid sequences homologous to PLCs of mammals and other organisms were amplified: one sequence from Botryotinia fuckeliana, one from Aspergillus nidulans, and three from Neurospora crassa. The molecular cloning and sequencing of a putative PLC gene (BCPLC1) from B. fuckeliana showed that it encoded a polypeptide containing X and Y domains, the two conserved regions found in all known PLCs. The hypothetical gene product of BCPLC1 was of delta type in its primary structural organization. The identification of three PLC genes in N. crassa shows that multiple PLC isozymes also occur in microbial eukaryotes.