Three independent mutations affecting N-acetylglucosaminidase in Dictyostelium discoideum were mapped by the parasexual system and found to lie on linkage group IV. These mutations as well as two others were found to be recessive and noncomplementing in heterozygous diploids. Thus they all appear to affect the nagA locus. Since two of the mutations give rise to thermolabile enzyme, this defines the structural gene for N-acetylglucosaminidase. The enzyme is a homodimer of a 68,000 dalton subunit and thus would be expected to be determined by a single locus. The expression of this gene is regulated by the stages of development; however, it should be mentioned that none of the mutations fell in a separate locus that might determine a specific positive regulatory proteins.