The factors that affect M/P (milk-to-plasma) ratios of several compounds were assessed in vitro using samples collected from four healthy lactating women. Three drugs were studied: diazepam, phenytoin, and propranolol. Serum and skim milk protein binding were determined by equilibrium dialysis at 37 degrees C. Skim-to-whole milk concentration ratios (S/M) were determined after incubation of whole milk at 37 degrees C for 1 h. Free fractions in serum and skim milk, respectively, were 0.013 and 0.334 for diazepam, 0.142 and 0.584 for phenytoin, and 0.107 and 0.699 for propranolol. The S/M values were 0.220, 0.727, and 0.610 for diazepam, phenytoin, and propranolol, respectively. An equation was derived which relates M/P to milk and plasma pH, unbound fractions of drug in skim milk and serum, and S/M. The M/P ratios calculated from in vitro data agreed with published in vivo M/P ratios. The results indicated that milk protein binding and fat partitioning can make a substantial contribution in determining M/P, and predictions of M/P made without considering these factors may be misleading.