The human population explosion has pushed many mammalian wildlife species to the brink of extinction. Conservationists are increasingly turning to captive breeding as a means of preserving the gene pool. We previously reported that serum immunoactive relaxin provided a reliable means of distinguishing between true and pseudopregnancy in domestic dogs, and this method has since been found to be a reliable indicator of true pregnancy in endangered Asian and African elephants and Sumatran rhinoceroses. Our canine relaxin radioimmunoassay (RIA) has now been adapted and validated to measure relaxin in the serum and urine of felids, including domestic and wild species. Moreover, a commercially available canine serum relaxin kit (Witness® Relaxin Kit; Synbiotics, San Diego, CA), has been adapted for reliable detection of relaxin in urine of some felid species. Our porcine relaxin RIA has also been utilized to investigate the role of relaxin in reproductive processes of the spotted hyena, a species in which the female fetuses are severely masculinized in utero. Indeed, this species might well now be extinct were it not for the timely secretion of relaxin to enable copulation and birth of young through the clitoris. Additional studies have suggested relaxin may be a useful marker of pregnancy in the northern fur seal and the maned wolf (the former species has been designated as “depleted” and the latter as “near threatened”). Given appropriate immunoassay reagents, relaxin determination in body fluids thus provides a powerful tool for conservationists and biologists investigating reproduction in a wide variety of endangered and exotic species.