Spt4p is a nonhistone protein of Saccharomyces cerevisiae that is believed to be required for normal chromatin structure and transcription. In this work we describe the isolation and analysis of a human gene, SUPT4H, that encodes a predicted protein 42% identical to Spt4p. When expressed in S. cerevisiae, SUPT4H complemented all spt4 mutant phenotypes. In human cells SUPT4H encodes a nuclear protein that is expressed in all tissues tested. In addition, hybridization analyses suggest that an SUPT4H-related gene is also present in mice. SUPT4H was localized to human chromosome 17 by PCR analysis of a human-rodent somatic cell hybrid panel. Thus, like other proteins that are components of or control the structure of chromatin, Spt4p appears to be conserved from S. cerevisiae to mammals.