In man, there are multiple forms of alkaline phosphatase encoded by at least three homologous genes: placental, intestinal, and liver/bone/kidney. This report describes the characterization of the human liver/bone/kidney alkaline phosphatase locus. The gene appears to exist as a single copy in the haploid genome and is comprised of 12 exons distributed over more than 50 kilobases. In liver, kidney, SAOS-2 human osteosarcoma cells, and cultured fibroblasts, there is a single major start for transcription situated about 25 nucleotides downstream of an A/T-rich motif. The promoter region is extremely G/C-rich, is relatively abundant in the dinucleotide CpG, and contains four copies of the consensus sequence for SP1 binding (GGGCGG). The liver/bone/kidney alkaline phosphatase gene is at least five times larger than the intestinal and placental alkaline phosphatase genes, mainly due to intron size differences. Intron-exon junctions occur at analogous positions in all three genes, but there is an extra non-coding exon at the 5' end of the liver/bone/kidney alkaline phosphatase gene. The relevance of our findings with respect to the evolution of the human alkaline phosphatase multigene family is discussed.