In the previous study in this series of studies concerning the role of macrophages in urinary tract infection, we attempted to detect macrophages in the urine of acute bacterial cystitis patients by nonspecific esterase staining of urinary sediment, however none of the leukocytes stained, probably because of cell damage caused by the urine and by centrifugation. In the present study, detection of macrophages in urine was again attempted, this time by prompt transfer of urinary leukocytes to culture medium after minimum centrifugation, 1 hr culture in a glass bottom chamber and non-specific staining of leukocytes adhering to glass. Macrophages in urine were detected by this method, and they comprised 5.9% of the adherent leukocytes, although macrophage spreading, which implies macrophage activation and is often seen in the early stage of nonbacterial prostatitis, was hardly ever observed. The percentages of adherent leukocytes were not correlated with urine osmolarity, probably because the effect of urine was minimized by prompt transfer of urinary leukocytes to culture medium after the urine samples had been collected. There have been quite few studies involving culture of urinary leukocytes in the past. Our simple techniques, such as prompt transfer of urinary leukocytes to culture medium after centrifuging with minimum gravity and for a minimum period of time, appear to be useful in the study of urinary leukocytes using other cells which appear in urinary tract infection, as well as cytokines and antibiotics, to clarify cellular mechanisms of defenses in urinary tract infection.