Ferredoxin is an iron-sulfur protein that serves as an electron transport intermediate for mitochondrial cytochromes P450 involved in steroid, vitamin D, and bile acid metabolism. We cloned and characterized the human ferredoxin gene family, which includes two expressed genes and two pseudogenes. Sequence analysis of this gene family revealed that it encodes only one protein product. The expressed genes were assigned to chromosome 11 and pseudogenes to chromosomes 20 and 21 by identifying single-copy probes from each gene segment and hybridizing them to DNA from rodent-human hybrid cells. The pseudogenes lacked introns and contained numerous mutations, including insertion, deletion, and substitution which rendered them inactive. They were 96% and 85% homologous to the expressed gene, yet they were only 78% homologous with each other. The intronless nature, higher diversity among themselves, and distinct chromosomal location of the pseudogenes suggests that they arose by independent, retroposon-mediated events.