Single-cell suspensions of granulocyte colony-stimulating factor (G- CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) cultured in alpha minimal essential medium (alphaMEM) containing 10% fetal bovine serum formed multicellular aggregates within 24 hours. In six separate experiments, formation of aggregates appeared to be dependent on cell density per surface area, so that 5.8 +/- 1.3 aggregates formed per 1 x 10(5) cells when G-PBMC were cultured at densities greater than or equal to 1 x 10(5) cells/cm2. The frequency of aggregate formation was less than 1 per 10(5) cells when G-PBMC were cultured at densities less than 1 x 10(5) cells/cm2. Once formed, aggregates became adherent within 72 hours, and then, over the course of 21 days, released CD3/CD4/CD25-positive cells into the supernatant. This T-cell production peaked between days 7 and 14, reaching a total of 1,269 +/- 125.9 cells released per aggregate by day 21. Between days 14 and 21, the aggregates also generated macroscopic clusters of adherent mononuclear and giant multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). At 4 weeks, the macroscopic foci coalesced into monolayers. Multinucleated TRAP-positive cells were distinguished from macrophage polykaryons by the absence of CD14 expression and the presence of osteoclast-specific membrane receptors for calcitonin and alphavbeta3-vitronectin. The osteoclast nature of these cells was further demonstrated by their ability to form resorption lacunae on dentine slices. Comparable osteoclast formation was not detected in cultures of normal marrow or normal nonmobilized peripheral blood.