Fecal 17β-estradiol and progestogens excretion was monitored in adult, female cheetahs (Acinonyx jubatus; n = 2), ZGG-12301 (born 3 April 1993), gonadotrophin treated and ZGT-3301, (born 19 August 1993), nontreated, for 120 days using commercially available plate enzyme immunoassay kits prepared for human serum or plasma. There were significant differences (P < 0.001) between baseline and peak concentrations of both hormone measures. Female ZGG-12301, which conceived, but this pregnancy resulted in an unobserved spontaneous abortion, showed no significant difference (P > 0.05) between baseline and gestation 17β-estradiol values; fecal 17β-estradiol excretion during pregnancy was statistically different (P < 0.001) from excretion during the nonpregnancy period. Baseline progestogen concentrations were different from pregnancy (P < 0.001) and postovulatory (P < 0.01) concentrations, and progestogen concentrations during pregnancy period were different (P < 0.001) from postovulatory concentrations. In the nontreated cheetah (ZGT-3301), basal and increased progestogen concentrations were statistically different (P < 0.01). On the basis of 17β-estradiol excretory patterns, duration of the estrous cycle (x̄ ± SEM) was 13.2 ± 2.2 days. These results suggest that the enzyme-linked immunosorbent methods reported in this study were capable of quantifying reproductive hormones in fecal extracts of cheetahs and could be a practical alternative to other enzyme-linked immunosorbent assays which require more complex procedures.