Serine/threonine protein phosphatase type 4 (PP4) belongs to a family of okadaic acid and microcystin-LR-sensitive protein phosphatases. In this study, we report the cloning and characterization of the human PP4 gene. The gene spans about 10 kb and includes one untranslated and eight translated exons. The 5' flanking region of the gene is rich in G and C (60.1%) and lacks TATA and CAAT boxes. Sequence analysis of the 5'-flanking region reveals potential binding sites for transcription factors SP1, AP1, AP2, and several gamma-IRE-CS sites. Two transcription initiation sites were mapped by ribonuclease protection analysis, one to 54 and the other to 84 bp upstream of the ATG initiation codon. PCR analysis of a human/rodent somatic cell hybrid panel maps PP4 to chromosome 16, and comparison of the PP4 gene structure with that of PP2A and PP1 suggests that PP4 is more closely related to PP2A than PP1.