Human prolactin from amniotic fluid, consisting of isohormones B and C (major), was radio-iodinated after storage of the hormone for 3 years at -70 degrees C, and yielded a Ferguson plot in polyacrylamide gel electrophoresis that was indistinguishable from the original except that the zones of isohormone B and C were fused. However, isohormones B and C of 125I-labelled human prolactin were separated on isoelectric focusing in polyacrylamide gel, using Ampholine carrier ampholytes (pI range 5-8), taurine (pI 5.1) as anolyte and beta-alanine (pI 6.9) as catholyte. After 20 h of electrofocusing at 0-4 degrees C, 1000 V, both isohormones reached constant pH (isoelectric) positions on the gel. The apparent isoelectric points of human prolactin B and C were 5.96. Micro-preparative gel electrofocusing followed by excision and re-electrofocusing of the gel slices containing human prolactin B and C, yielded zones of homogeneous isohormones B and C.