Volumetrically distinct subpopulations of peripheral blood monocytes, termed M1, M2, and M3, were identified in healthy normal adults and children. Because normal neonates have abnormal monocyte chemotaxis, it was determined whether monocyte subpopulations have different chemotactic capabilities and, if so, whether chemotactically active subpopulations were quantitatively deficient in neonates. Chemotaxis tests with zymosan-activated normal human serum as the chemoattractant and purified monocyte subpopulations revealed that large M3 monocytes were capable of significantly more directed migration than were small M1 and M2 monocytes. Volumetric analysis of monocytes from normal newborns rather than demonstrating an absence of M3 cells revealed that these cells were the predominant monocyte subpopulation. Therefore, we conclude that the impaired chemotactic ability of newborn monocytes is due to a functional rather than quantitative deficiency of M3 cells.