Tritiated 1-(2, 4-dichlorobenzyl)-1H-indazole-3-carboxylic acid ([3H]AF 1890) was localized within rat testis using freeze-drying autoradiographic techniques. Ligated seminiferous tubule preparations were exposed in vitro to 80 microCi of [3H]AF 1890 in the absence or presence of excess unlabeled AF 1890 for 30 min at 31 degrees C. Autoradiographs showed a generalized distribution of silver grains over the seminiferous epithelium, in the tubular lumen, and in the interstitial spaces. In addition, clusters of grains were concentrated over the seminiferous epithelium in regions of spermatid heads and tails and in the basal portion of the Sertoli cell cytoplasm, and over Leydig cells in the interstitial space. The generalized distribution was reduced and the grain clusters eliminated by incubation in an excess of unlabeled AF 1890. [3H]inulin was used to assess the effectiveness of the tubular ligation because inulin does not normally penetrate the blood-testis barrier. This extracellular marker was not localized in the tubular lumen or in the seminiferous epithelium. Therefore, the labeling observed with [3H]AF 1890 indicates that this compound crosses the blood-testis barrier.