Extracts from 12 pituitaries were applied to preparative immunoaffinity chromatography columns containing between 500 mg and 800 mg of clonal antiprolactin IgG1. Extracts were cycled through the column and subsequently washed from the affinity matrix. Total prolactin was eluted from the affinity matrix by a 0.1 M glycine, pH 2.8. The total prolactin was neutralized and dialyzed into 20 mM Bis-Tris (pH 7.0) and applied to a weak anion exchange column. Nonphosphorylated prolactin was eluted with 0.5 M NaCl in Bis-Tris buffer. Phosphorylated prolactin was eluted with 20% acetic acid. Yields averaged 19.6 mg and 11.7 mg for native and phosphorylated prolactin (n = 14 runs), respectively. The molecular weights of the prolactins were similar to prolactin standards. The amino acid compositions of the two prolactin variants were similar. Phosphate was associated predominantly with the phosphorylated prolactin. Native prolactin was biologically active in the Nb2 rat lymphoma bioassay, while the phosphorylated prolactin was without activity in this bioassay.