Three bioassays are widely employed for the measurement of PTH‐like adenylate cyclase‐stimulating factors (ACSFs) drived from tumors associated with humoral hypercalcemia of malignancy. These include renal cortical adenylate cyclase (RAC) assays, rat osteosarcoma (ROS) adenylate cyclase assays, and fetal bone resorption (FBR) assays. A previous study has suggested that the potency of one human tumor‐derived ACSF, expressed in PTH equivalents, was 30‐fold higher in the ROS assay than in the RAC assay, but no study has directly compared all three bioassays using a single PTH standard and a single ACSF preparation. We compared one partially purified ACSF preparation to a single lot of bPTH 1–34 in all three bioassays. The results indicate that the relative potency of this ACSF as compared to the PTH standard varied with the assay employed, with the ROS assay yielding a specific activity estimate 47.5‐fold higher than the RAC, and the FBR 6.7‐fold higher than the RAC but 7.1‐fold lower than the ROS. These findings support the possibility that distinct subpopulations of PTH receptors exist on different PTH target tissues. Further, they underscore the importance of bioassay choice when estimating the specific activity of tumor‐derived ACSF preparations.