A simple and practical radioimmunoassy (RIA) for pregnanediol without any pretreatment of urine specimens such as hydrolysis and/or extraction was developed. Antisera were produced by immunizing rabbits with pregnanediol-3-glucuronide (P-diol-3-G)-BSA conjugates and P-diol-3-hemisuccinate-BSA conjugates. The specificities of the antisera were examined by their cross-reactivities with related steroids. Antiserum to P-diol-3-G-BSA was significantly cross-reacted with 20 alpha-OH-P(201%), P-diol(100%), P -diol-3-G (77.3%, where 119% as free P-diol), to a lesser extent with 20 beta-OH-P(17.8%), and did not react with other steroids. Antiserum to P-diol-3-hemisuccinate-BSA, on the other hand, was capable of reacting with P-diol(100%), P-diol-3-G(46.9%), 20 alpha-OH-P(75.2%) and 20beta-OH-P (8.97%), respectively. Antiserum generated against P-diol-3-G-BSA was almost twice as high in its avidity for P-diol-3-G as compared with antiserum raised against P-diol-3-hemisuccinate-BSA. Antiserum to 20alpha-OH-P-3-carboxy-methyl oxime-BSA conjugate was only reactive with 20alpha-OH-P and not with P-diol or P-diol-3-G. From the data presented here, antiserum to P-diol-3-G-BSA with high specificity toward urinary P-diol-3-G was used for the present assays. Ammonium sulfate precipitation was employed for the separation of antibody-bound and free tracer hormones. A direct method in which simply diluted urine was directly applied to RIA and the enzymatic hydrolysis method which requires extraction of P-diol with ether following hydrolysis of urine by beta-glucuronidase were both examined in the present RIA on the same specimens which contained known amounts of 3H-P-diol-3-G. Values obtained by the direct method were in good correlation with those determined by the hydrolysis method (r=0.994 p less than 0.01, Y=1.07X + 0.30). The working range of the assay was from 50pg to 5ng of P-diol per tube, and intra- and interassay precisions were 9.8, 12.5 and 12.9, 16.7%, respectively. Determination of radioimmunoassayable P-diol-3-G in urine was also in good correlation with the values obtained by the currently used analytical procedure, the Klopper-Kambegawa method.