KRM-1648 is a new benzoxazinorifamycin with activity in vitro and in vivo against organisms of the Mycobacterium avium complex. We investigated the ability of 14C-KRM-1648 to concentrate within human monocyte-derived macrophages in vitro. KRM-1648 is rapidly taken up by uninfected macrophages, with 90% of the initial concentration added to the monolayer found within macrophages by 1 h and approximately 80% at 2 h. Comparable results were obtained in assays using macrophages that have been infected with an AIDS-related strain of M. avium for 24 h. In contrast, macrophages infected with M. avium for 3 days, showed an impaired ability to concentrate KRM-1648, primarily because of a significant efflux of the antibiotic (intracellular concentration of 86% of the available drug was present within macrophages at 1 h vs. 47% at 2 h). Daily administrations of KRM-1648 to a macrophage monolayer for 3 consecutive days resulted in significant accumulation of the drug within phagocytic cells. Although the efflux was greater in M. avium-infected macrophages than in uninfected cells, consecutive administration of KRM-1648 led to a total intracellular accumulation of drug that exceeded the initial level and appeared to continue to accumulate. The ability of KRM-1648 to rapidly accumulate in human macrophages, including M. avium-infected cells, may explain, in part, the improved therapeutic effectiveness in animal models against M. avium and M. tuberculosis.