The six members of the human prothymosin alpha gene family have been cloned and sequenced. One gene (PTMA) contains introns and appears to be the source of all isolated prothymosin alpha cDNAs. The remaining five genes are processed pseudogenes. Four of them have consensus TATA elements upstream of sequences nearly identical to the transcriptional start region of the intron-containing gene. Those four genes also contain open reading frames coding for proteins closely related to prothymosin alpha. In two of the pseudogenes, PTMAP2 and 5, the encoded proteins differ from the product of the parental gene at only two and four locations, respectively. The fifth pseudogene (PTMAP1) encodes a different protein owing to an upstream translational initiation start site and multiple deletions and insertions. Because the potential for expression exists in this system, a search for pseudogenomic transcripts was undertaken using the polymerase chain reaction to amplify reverse transcripts of mRNAs from many human tissues and bulk DNA from several human cDNA libraries. Evidence for pseudogenomic transcripts was not obtained. Therefore, we conclude that the human prothymosin alpha gene family contains only one functional gene.