Osteoclasts resorb bone through transient rearrangement of their cytoskeletons to create a polarized phenotype in which an apical ruffled membrane is surrounded by a ring of F-actin that creates a tight seal against bone substrate. This process, coupled with the capacity for rapid motility, necessitates the presence of a dynamic, multi-functional actin cytoskeleton. Tropomyosins are a large class of actin-binding proteins that can regulate microfilament stability and organization by recruiting other regulatory proteins to actin, or alternately, by inhibiting their binding. Tropomyosins are expressed from four distinct genes (alpha, beta, gamma, and delta) that are alternately spliced to produce over forty isoforms. In recent years, it has become clear that nonmuscle isoforms of tropomyosin may be differentially distributed among intracellular pools of F-actin possessing different functions. Here we have used Western analysis and immunocytochemistry coupled with confocal microscopy to identify the isoforms of tropomyosin expressed by osteoclasts, as well as their distributions within cells. Osteoclasts express at least seven isoforms with markedly different distributions. The products of the alpha gene (Tm-2, -3, and -5a/5b) are up-regulated during osteoclastogenesis, indicating potential cell-specific functions. Some isoforms (Tm-5a/5b, Tm-4) are specifically enriched within and around osteoclast attachment structures, the sealing zone and podosomes, whereas others are more abundant in internal regions of the cell. This compartmentalization of tropomyosins to specific actin structures within osteoclasts is likely to play a critical role in determining the dynamic properties of the actin cytoskeleton and thus osteoclast activity.