A specific and sensitive radioimmunoassay method for progesterone and its application to clinical practice are described. Antiserum of progesterone was prepared in rabbits by repeated subcutaneous injections of 11 α-hydroxyprogesterone-bovine serum albumin conjugate emulsified in complete Freund's adjuvant. The antiserum was so specific that the chromatographic purification could be omitted for sample preparation. A conjugate of 11 α-hydroxyprogesterone with tyrosine methyl ester was also prepared, and radioiodinated by the chloramine-T method. Immunoreactivity of the labeled progesterone was stable at 4°C for up to 40 days. Snsitivity of the assay was 0.05 ng/tube. The intra- and inter- assay coefficient variations were 7.0% and 14.4%, respectively. There was a linear correlation (r=0.95) between the amounts of progesterone added and the values determined by the radioimmunoassay method. The mean recovery rate was 86.4%. Serum progesterone levels in volunteers and infertile patients with histological diagnosis of normal luteal function are statistically higher in mid-luteal phase than any progesterone levels in patients with histological diagnosis of luteal insufficiency. This indicates that any single determination of serum progesterone levels in mid-luteal phase might be useful in evaluating corpus luteumu function.