Prolactin-binding sites were determined in 759 human breast tumor biopsy specimens. Ovine prolactin (oPRL) was used in the binding assay of 569 tumors with 13% of the tumors showing a positive content with more than 1% specific binding. Human prolactin (hPRL) was utilized for 343 tumors with 36% of the tumors binding 1% or greater of the added radioactivity. When human growth hormone (hGH) was used as the labeled ligand in 95 tumors, only 2% of the tumors specifically bound more than 1% of the hGH. A total of 153 tumors were assayed simultaneously with the use of labeled hPRL and oPRL. Whereas 10.4% of the tumors showed the presence of prolactin-binding sites (greater than 1% specific binding) when oPRL was used, an approximately threefold increase (29.4%) in tumors possessing prolactin receptors was revealed when hPRL was used as the labeled ligand. Therefore, hPRL is the preferred ligand for the assessment of prolactin receptor levels in human breast cancer biopsy specimens.