New derivatives of progesterone and aldosterone were synthesized and functionally tested with commercially available antibodies. The covalent labelling of antibodies specific for aldosterone and progesterone was detected by SDS/PAGE analysis and subsequent autoradiography after using 3-(O-carboxymethyl)-oximino-(3-[125I]iodo-4-azidosalicylamidobu tylamine) derivatives of aldosterone and progesterone, respectively, as photoactivatable radioligands. Labelling was not observed in the presence of an excess of the unlabelled steroid. Aldosterone was labelled with biotin and used as a tracer in a time-resolved fluorescence immunoassay. The nonradioactive tracer is highly selective for its antibody-binding site, with almost no detectable cross-reactivity for other steroids. Biotin-labelled progesterone was immobilized by avidin-agarose and used for affinity chromatography. This yielded a more than 20-fold enrichment of an anti-progesterone polyclonal antibody. These results demonstrate that derivatives of steroids are particularly useful for the development of nonradioactive assays for the determination of natural steroids and may be also useful for the detection of specific binding sites in biological material such as plasma membranes.