Non-invasive monitoring of the reproductive function in animals is possible by measuring steroid metabolites in the faeces. After infusion of C-14-steroids, differences between species concerning both the percentage and the delay of faecal excretion can be observed. In domestic livestock almost all faecal C-14-metabolites are found in an unconjugated form. The aim of this study was to characterize the main metabolites present in faecal samples of sheep, ponies and pigs after infusion of C-14-progesterone and to evaluate different extraction procedures. This should help to standardize and improve faecal steroid analysis. Faeces containing peak radioactivity were suspended in methanol/water and Sep-Pak(R) C-18 cartridges combined with Sephadex(R) LH-20 columns were applied to clean up the samples. Fractions were subjected to straight phase high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC). Metabolites were classified by comparison with the elution patterns of steroid reference standards and by testing their immunoreactivity in different enzyme-immunoassays (EIAs). The results revealed considerable differences in the pattern of faecal progesterone metabolites between species. A great number of metabolites (mainly mono- and di-hydroxylated pregnanes as well as pregnanediones) was found and progesterone barely, if at all. In sheep, pregnanes containing a 20 alpha-hydroxy (OH) group dominated (about 45%), whereas in ponies metabolites with a 20 beta-OH group (about 44%; mainly one 5 alpha-pregnanediol), and in pigs progestagens with a 20-oxo group (46-58%) predominated. Suspending the faeces with a high percentage of methanol, ethanol or iso-propanol yielded the best recoveries and enabled a practicable extraction, without an evaporation step. As a wide range of metabolites was formed, group-specific immunoassays using antibodies with high cross-reactions with pregnanes sharing a similar C-20 group should be used for measuring faecal metabolites. To verify the data of the C-14-experiments, various progesterone metabolites were measured in faecal samples of superovulated ewes. In accordance with the results of the characterized progesterone metabolites, an EIA measuring 20 alpha-OH-pregnanes proved most suitable for non-invasive monitoring of the corpus luteum function in sheep.