Sequence-tagged sites (STSs) are versatile chromosomal markers for a variety of genome mapping efforts. In this report, we describe a randomly generated STS (323F4) from human chromosome 16 genomic DNA that has 90.0% sequence identity to the type I human inosine-5'-monophosphate dehydrogenase (IMPDH1) gene and 72% identity to the type II human inosine-5'-monophosphate dehydrogenase (IMPDH2) gene. Additional sequencing by primer walking has provided a total of 1380 bp of the human chromosome 16 sequence. The IMPDH-like sequence 323F4 was regionally localized by PCR analysis of a panel of somatic cell hybrids containing different portions of human chromosome 16 to 16p13.3-13.12, between the breakpoints found in hybrids CY196/CY197 and CY198. This regional mapping assignment was further refined to subband 16p13.13 by high-resolution fluorescence in situ hybridization using cosmid 323F4 as a probe. We conclude that a third, previously undescribed IMPDH locus, termed IMPDHL1, exists at human chromosome 16p13.13.