In order to identify amino acids directly involved in progesterone binding to rabbit uteroglobin we have mutated Phe 6, Tyr 21 and Thr 60 by site-directed mutagenesis of the uteroglobin cDNA. These residues have been postulated previously to participate in progesterone binding. High-level expression of the mutated uteroglobin cDNAs in Escherichia coli yields recombinant protein mutants that, like natural uteroglobin, form stable dimers, suggesting that the tertiary structure of the protein has not been altered. Substitution of Phe 6 by Ser or Ala does not change the progesterone binding characteristics. In contrast, replacement of Tyr 21 by Phe or Ala, drastically decreases progesterone binding. In addition, replacement of Thr 60 by Ala reduces the affinity for progesterone by a factor of three. These data suggest a direct interaction of progesterone with these two amino acids and support the idea of direct hydrogen bonding of the carbonyl (C3 and C20) of progesterone with the hydroxyl groups of Tyr 21 and Thr 60, respectively.