The possible existence of forms of rat PRL with different bioassay (BA)/RIA activity ratios was investigated. Anterior pituitary tissue and medium in which explants of rat adenohypophyses had been incubated were processed by polyacrylamide disc gel electrophoresis using a Tris-glycine buffer. The gel columns were cut into five segments and eluates from these were tested for PRL by BA and by RIA. Most of the BA- and RIA-detectable PRL in tissue and medium was found in the same gel region to which purified rat PRL (RP-1) migrated. The BA/RIA ratio of this major form of PRL was 2.2-4.0. Other more slowly migrating forms of PRL were found in the regions where albumin and growth hormone migrate. The BA/RIA activity ratios of these forms varied depending on the sample processed (e.g., tissue vs. medium; sex and/or age of the adenohypophyseal donor; duration of incubation) but were generally considerably higher than those of the respective major forms. PRL that migrated faster than the major form was also detected by the RIA in several cases, but in only one experiment (with 4-day culture medium) was bioassayable PRL present in this more anodal region. In this case the BA/RIA ratio was very high (78.6). These results indicate that forms of rat PRL with different BA/RIA activity ratios exist within and are secreted by rat adenohypophyses.