The formation of rosettes between human blood monocyte-derived macrophages and lymphocytes (MLR) in samples harvested from total leukocyte (TL) cell cultures, was confirmed. Experiments with leukocytes obtained from human blood of healthy individuals (n = 17) and prepared under various conditions, were performed. Cytopreparations of each experiment were used for classical staining procedures or for immunohistochemical methods with monoclonal lymphocyte surface markers. Recently obtained blood leukocytes were unable to form MLR, whereas cultured samples of the same cells started to form MLR 15 hr after culturing. At that time, the number of MLR in pelleted samples was 1.18%, reaching a peak of 15.7% at 120 hr of culturing. In cultured but nonpelleted samples, only a few MLR were formed. With monoclonal antibodies, the lymphocytes forming MLR reacted mainly as CD4 positive and much less as CD8 (the ratio was 18:1). Monocyte-derived macrophages were able to form MLR when they underwent transformation into macrophages. The finding that the lymphocytes involved are T-cells, mainly CD4 positive, suggests that in the cell-cell interaction, macrophages could be presenting antigens to the lymphocytes. Besides, because the highest number of MLR occurred in TL samples, whereas few rosettes were formed in the mononuclear cell samples, the existence of some particular mechanism(s) acting on TL samples is suggested.