Three-color flow cytometry was used to assess the immunophenotypic characteristics of normal cord blood monocytes after labeling with a variety of antibodies against myeloid/monocyte-specific markers. Monocytes both in cord blood and in peripheral blood from normal adults were defined in a backgating procedure as cells with the light-scattering characteristics of monocytes that also expressed CD14. The percentage of monocytes, defined in this fashion, that also displayed CD4 receptors was significantly lower in cord blood (mean +/- SD = 29.3 +/- 13.9%) than in peripheral blood from normal adult controls (mean +/- SD = 68.9 +/- 13%) (P < 0.005). Similarly, HLA-DR expression was found on only 86 +/- 6.6% of monocytes in cord blood but on 99 +/- 1% of monocytes in adults (P < 0.005). The percentage of monocytes displaying CD16 receptors in cord blood did not show any significant difference in comparison with adult monocytes. When coexpression of CD14, CD16, and CD4 was assessed, cord blood showed a predominant population of monocytes bearing the phenotype CD14+/CD16-/CD4-. Similarly, approximately 10% of CD14+ monocytes in cord blood expressed neither CD4 nor HLA-DR. Cytochemically, monocytes from cord blood revealed intense granular staining for PAS and marginal or absent staining for nonspecific esterase (NSE). These results raise the possibility that reduced expression of CD4 and HLA-DR receptors on cord blood monocytes may contribute to their impaired immune response. Additionally, the high percentage of CD14+/CD16-/CD4- cells in cord blood suggests that these cells may represent a phenotypically immature population of monocytes. Likewise, the unusual cytochemical staining patterns suggest that these cells are biochemically immature as well.