Spontaneous mutants resistant to nalidixic acid (NAL) were isolated from the petite negative yeast Schizosaccharomyces pombe (S. pombe). One of these mutants, resistant to 200 micrograms/ml NAL, nalr-Y13, was characterized both genetically and biochemically. The extrachromosomal inheritance of this mutation was demonstrated both by mitotic segregation and by mitotic haploidization analysis. In the wild-type, NAL at a concentration of 100 micrograms/ml almost completely inhibits incorporation of [14C]adenine in total DNA as well as in mitochondrial DNA. In the NAL-resistant mutant both total DNA synthesis and mitochondrial DNA synthesis were resistant to the drug. These results are discussed in view of previously published findings on the close interaction between the two DNA synthesizing systems in S. pombe.