During the first 4 neonatal weeks, serial total fatty acid and individual fatty acid balance studies were completed in 35 healthy premature infants with appropriate weight for gestational age. Infants weighed <1500 gm at birth, were descriptively similar, and were receiving similar volumes of either preterm mothers' milk (PTM) or formula (S-M-A 20). Total fatty acid and major fatty acid contents of the two feeding regimens were similar. Total fecal output and total fatty acid excretion were higher, whereas subsequent total fat absorption and coefficient of absorption were significantly lower, in the group fed S-M-A 20 (P<0.001). Administration of oral calcium supplements fed as calcium lactate (1.5 to 2.0 mmol/kg/day) decreased total fatty acid absorption in both the PTM (P<0.01) and S-M-A 20 (P<0.001) groups. Furthermore, the effect of feeding (P<0.0001) and oral calcium (P<0.001) independently influenced coefficients of absorption for major fatty acids fed (C12:0, C14:0, C16:0, C18:0, and C18:1). Although the main indices for growth were similar in both feeding groups, infants with inefficient rates of total fatty acid absorption attained slower rates of weight gain and increased skinfold thickness. We conclude that oral calcium supplements significantly alter the efficiency of lipid absorption in enterally fed preterm infants.