We describe the isolation of a nonhematopoietic (CD45-, CD34-, SH2+, SH3+, Thy-1+, CD44+) human umbilical cord perivascular (HUCPV) cell population. Each HUCPV cell harvest (2-5 x 10(6), depending on the length of cord available) gave rise to a morphologically homogeneous fibroblastic cell population, which expressed alpha-actin, desmin, vimentin, and 3G5 (a pericyte marker) in culture. We determined the colony-forming unit-fibro-blast (CFU-F) frequency of primary HUCPV cells to be 1:333 and the doubling time, which was 60 hours at passage 0 (P0), decreased to 20 hours at P2. This resulted in a significant cell expansion, producing over 10(10) HUCPV cells within 30 days of culture. Furthermore, HUCPV cells cultured in nonosteogenic conditions contained a subpopulation that exhibited a functional osteogenic phenotype and elaborated bone nodules. The frequency of this CFU-osteogenic subpopulation at P1 was 2.6/10(5) CFU-F, which increased to 7.5/10(5) CFU-F at P2. Addition of osteogenic supplements to the culture medium resulted in these frequencies increasing to 1.2/10(4) and 1.3/10(4) CFU-F, respectively, for P1 and P2. CFU-O were not seen at P0 in either osteogenic or non-osteogenic culture conditions, but P0 HUCPV cells did contain a 20% subpopulation that presented neither class I nor class II cell-surface major histocompatibility complexes (MHC-/-). This population increased to 95% following passage and cryopreservation (P5). We conclude that, due to their rapid doubling time, high frequencies of CFU-F and CFU-O, and high MHC-/- phenotype, HUCPV cells represent a significant source of cells for allogeneic mesenchymal cell-based therapies.