The human prostate is a complex glandular organ with functional development under hormonal regulation. Diseases of the prostate result in significant morbidity and mortality in the form of benign prostatic hypertrophy and prostate adenocarcinoma. The characterization of the molecular framework of the human prostate at the level of expressed genes will facilitate the understanding of normal and pathological prostate biology. The purposes of this study were to acquire an initial assessment of the qualitative and quantitative diversity of gene expression in the normal human prostate and to determine the extent that genes with prostate-restricted expression can be assessed using an expressed sequence tag approach. We have constructed a directional cDNA library from normal adult human prostate tissue and partially sequenced the 5' end of 1168 randomly selected cDNA clones, resulting in more than 400 kb of DNA sequence. Homology searches of the sequenced cDNAs against the GenBank and dbEST databases revealed that 43% of the sequences are identical to human genes whose functions are known, 5% are similar but not identical to known genes in humans or lower organisms, 5% match the mitochondrial genome, 9% are composed of interspersed DNA repeats, 30% are homologous to sequences in the dbEST database without a described function, and 6% are novel sequences. A total of 780 distinct species were identified. In addition to the 74 novel transcripts, 4 genes, prostate-specific antigen (PSA), prostate secretory protein (PSP), prostate acid phosphatase (PAP), and human glandular kallekrein 2 (HK2), have no homologous sequences in the databases that originate from sources other than prostate and thus may represent genes with prostate-restricted expression. Sequences matching PSA, PSP, and PAP each accounted for > 1% of the total ESTs and represent highly abundant transcripts, correlating with the abundance of these proteins in the prostate gland. No novel transcripts were represented by more than one EST and thus are expressed at levels much lower than the known prostate-specific genes.