The sex of three short-beaked echidnas (Tachyglossus aculeatus), part of a captive colony in which no successful births have occurred, was determined by measuring levels of faecal sex steroids over a period of two months. Levels of faecal oestradiol-17beta were found not to differ between male and female short-beaked echidnas. However, measurement of the oestradiol : androgen ratio (E : A ratio) and androgen concentrations successfully identified one male and two female echidnas. Mean E : A ratios for the two females were 18 +/- 6 and 23 +/- 8, compared with 1.3 +/- 0.6 for the male. The mean androgen concentration for the male was 10.1 +/- 2.3 ng g(-1) dried faeces, and 1.7 +/- 0.5 and 2.2 +/- 0.6 ng g(-1) dried faeces for the two females. The E : A ratio allowed correct identification of the sex of the echidnas 80% of the time, whereas the androgen concentration correctly identified the sex 75% of the time but was reliable only during the breeding season. Both the male and female echidnas showed evidence of gonadal activity; lack of breeding in captivity was therefore not due to all animals being of the same sex or lacking reproductive activity. The study has demonstrated that faecal steroid analysis is an accurate method for sex determination in the short-beaked echidna and further monitoring of these hormones may elucidate many of the unknown aspects of their reproductive biology.