The Aspergillus nidulans 3-phosphoglycerate kinase gene (PGK) has been isolated from a phage lambda genomic library, using the equivalent yeast gene as a hybridization probe. The location of the PGK gene within the cloned DNA has been physically mapped. The DNA sequence of a small region of the putative PGK has been determined and found to code for amino acids corresponding to the N-terminal end of the PGK protein. In contrast to the yeast PGK gene the Aspergillus gene contains a 57 base pair intron occurring between the coding sequences for amino acid 22 and 23. A DNA fragment encompassing the PGK gene was shown to hybridize a 1,700 base poly(A) mRNA, sufficient to encode the PGK polypeptide.