Recent results showing that the binding characteristics of 33 steroids for human membrane progesterone receptor alpha (hu-mPRalpha) differ from those for the nuclear progesterone receptor (nPR) suggest that hu-mPRalpha-specific agonists can be identified for investigating its physiological functions. The binding affinities of an additional 21 steroids for hu-mPRalpha were determined to explore the structure-activity relationships in more detail and to identify potent, specific mPRalpha agonists. Four synthetic progesterone derivatives with methyl or methylene groups on positions 18 or 19, 18a-methylprogesterone (18-CH(3)P4, Org OE 64-0), 13-ethenyl-18-norprogesterone (18-CH(2)P4, Org 33663-0), 19a-methylprogesterone (19-CH(3)P4, Org OD 13-0) and 10-ethenyl-19-norprogesterone (19-CH(2)P4, Org OD 02-0), showed similar or higher affinities than progesterone for hu-mPRalpha and displayed mPRalpha agonist activities in G-protein and MAP kinase activation assays. All four steroids also bound to the nPR in cytosolic fractions of MCF-7 cells. However, two compounds, 19-CH(2)P4 and 19-CH(3)P4, showed no nPR agonist activity in a nPR reporter assay and therefore are selective mPRalpha agonists suitable for physiological investigations. The structure-binding relationships of the combined series of 54 steroids for hu-mPRalpha deviated strikingly from those of a published set of 60 3-keto or 3-desoxy steroids for nPR. Close correlations were observed between the receptor binding affinities of the steroids and their physicochemical properties calculated by comparative molecular field analysis (CoMFA) for both hu-mPRalpha and nPR. A comparison of the CoMFA field graphs for the two receptors revealed several differences in the structural features required for binding to hu-mPRalpha and nPR which could be exploited to develop additional mPR-specific ligands.