This study was undertaken to determine if PRL variants differing in structural characteristics as well as biological activity and immunoreactivity are present in human milk and to assess whether the physiological state of the mammary gland (premature initiation of lactation, progression of lactation) influences the forms or activity of human milk-borne PRL. Human milk samples were collected in early (day 1-5) or mature lactation (1-4 months) from women delivering term (T) or preterm (PT) infants. Milk was fractionated on Sephadex G-100, Concanavalin A-Sepharose, or Phosphogel to estimate molecular weight (MW), degree of glycosylated PRL(G), and degree of phosphorylated (P) PRL, respectively. Prolactin-like bioactivity (B) was measured by the PRL-dependent proliferation of the Nb-2 lymphoma cell and PRL-like immunoreactivity (I) by radioimmunoassay (RIA) using NIDDK reagents. Milk obtained from mothers of T infants contained significantly greater B than I-PRL at each stage of lactation (early: B = 132 +/- 13.9 P.E./ml, I = 83.43 +/- 12 P.E./ml, mature: B = 41.74 +/- 8.9 P.E./ml, I = 27.19 +/- 5.5 P.E./ml; P < 0.01-0.001). PRL-like bioactivity was greatest in early milk produced by mothers of T infants and declined as lactation progressed. No differences in B or I-PRL were evident for milk collected from mothers of preterm infants at any stage of lactation. In early lactation the PRL-like bioactivity of milk collected from mothers of T infants was significantly greater than in unfractionated milk collected from mothers of PT infants (P < 0.05). The number of PRL variants present in milk was characteristic of the stage of lactation and gestation (T early > PT early; T early > T mature). Early milk from mothers of T infants contained an average of 5 bioactive PRL variants, early milk from mothers of PT infants an average of 4 bioactive variants, and mature milk from mothers of T infants an average of 3 bioactive variants. The distribution of molecular weights associated with PRL variants in milk infranatant was variable among subjects within a specific stage of lactation/gestation. However, the representative range of molecular weights of PRL variants identified in milk sample was similar between groups (T early: < 8, 16, 20, 24, 25-28, 32, 42, > 66; PT early: 16, 20, 23, 25-28, 32, > 66; T mature: 20, 25-28, 32, 35, > 66).(ABSTRACT TRUNCATED AT 400 WORDS)