Lactoferrin (LF) was isolated from human milk by serial procedures of 45% ammonium sulfate precipitation, CM Sephadex C-50 ion-exchange chromatography, Sephadex G-100 gel filtration, and Cu-affinity chromatography, in which 59Fe lactoferrin was used as the tracer. The recovery of LF from human milk was 3.3%. LF from human milk was a single component having a molecular weight of 78k on SDS-PAGE, and showed pI 8.02 by isoelectric focusing on slab gel. The LF concentration was measured by rocket immunoelectrophoretic assay using anti-human LF antiserum in human colostrum and milk, from 1 to 60 days after parturition (125 samples). The LF concentrations in colostrum (1-3 days of puerperium, n = 35), the transitional milk (4-7 days, n = 60), and mature milk (20-60 days, n = 30) were 6.7 +/- 0.7, 3.7 +/- 0.1, and 2.6 +/- 0.4 (mean +/- SEM) g/liter, respectively. Both the LF and total protein (TP) concentrations showed significantly inverse correlations with the days after parturition (p less than 0.001). The lactoferrin/total protein ratio (LF/TP) in the mature milk (16.1 +/- 1.4%) was significantly less than that in the colostrum (20.4 +/- 1.2%, p less than 0.05) and the transitional milk (21.4 +/- 0.9%, p less than 0.05). Furthermore, iron concentration (Fe) in human milk was also measured by the internal standard technique of the spiked method on atomic absorption, and the lactoferrin iron saturation (LS%) was calculated. Neither Fe nor LS% had significant difference among these three stages of the lactation. The means (n = 125) of Fe and LS% were 60.6 +/- 5.4 micrograms/100 ml and 11.8 +/- 1.1%, respectively. However, significant correlation was observed between LF and Fe (p less than 0.005) or between LF/TP and both of Fe (p less than 0.05) and TP (p less than 0.001) in the mature milk. These results suggest that the mechanism stimulating the synthesis and secretion of LF is different from those of other proteins and LF can play variable roles in iron nutrition of babies at the different stages of lactation.