Huntingtin, the protein product of the Huntington's disease (HD) gene, is expressed with an expanded polyglutamine domain in the brain and in nonneuronal tissues in patients with HD. Huntingtin-associated protein 1 (HAP-1), a brain-enriched protein, interacts preferentially with mutant huntingtin and thus may be important in HD pathogenesis. The function of HAP-1 is unknown, but recent evidence supports a role in microtubule-dependent organelle transport. We examined the subcellular localization of HAP-1 with an antibody made against the NH 2 -terminus of the protein. In immunoblot assays of mouse brain and immortalized striatal neurons, HAP-1 subtypes A and B migrated together at about 68 kD and separately at 95 kD and 110 kD, respectively. In dividing clonal striatal cells, HAP-1 localized to the mitotic spindle apparatus, especially at spindle poles and on vesicles and microtubules of the spindle body. Postmitotic striatal neurons had punctate HAP-1 labeling throughout the cytoplasm. Western blot analysis of protein extracts obtained after subcellular fractionation and differential centrifugation of the clonal striatal cells showed that HAP-1B was preferentially enriched in membrane fractions. Electron microscopic study of adult mouse basal forebrain and striatum showed HAP-1 localized to membrane-bound organelles including large endosomes, tubulovesicular structures, and budding vesicles in neurons. HAP-1 was also strongly associated with an unusual large ''dense'' organelle. Microtubules were labeled in dendrites and axonal fibers. Results support a role for HAP-1 in vesicle trafficking and organelle movement in mitotic cells and differentiated neurons and implicate HAP-1B as the predominant molecular subtype associated with vesicle membranes in striatal neurons. J.