A major difficulty in sea turtle conservation is the inability to nonlethally and noninvasively identify the sex of hatching sea turtles. Traditional sexing techniques such as plasma sex steroid quantification cannot be applied to hatchlings without sacrificing the hatchlings or utilizing invasive procedure. This paper presents a technique for sexing hatchling sea turtles by analysis of sex steroid concentrations in egg chorioallantoic/amniotic fluid (CAF). Metabolites of estradiol-17 beta (E) and testosterone (T) in CAF are best expressed as an index or E:T ratio. Chorioallantoic/amniotic fluid E:T ratios for males (0.5 +/- 0.1) were significantly lower than those for females (2.2 +/- 0.3). When separated by utilizing an E:T ratio of 1.25 as the determinant index value, 27 of 28 hatchlings were designated correctly as males (E:T < 1.25) or females (E:T > or = 1.25). Sex was verified for all hatchlings by gonadal histology. This study shows significant concentrations of T and E metabolites in CAF and plasma of hatchling loggerhead turtles and illustrates the use of a nonlethal, noninvasive method for determining sex, which could be potentially utilized for other endangered reptile and avian species.