Differentiation and growth of chondrocytes in fetal growth plates of vertebrate long bones and ribs appear to occur in a gradual, continuous manner between the resting zone through the proliferation zone, maturation zone, and upper and lower hypertrophic zones, with a continuous increase in cell size up to 10-fold of the volume of a resting chondrocyte. Here we provide evidence, however, that after centrifugation through a continuous Percoll gradient growth plate chondrocytes separate into four distinct cell populations (B1 to B4) which differ markedly in density, size, and gene expression. These populations collect in the absence of any phase borders in the gradient which might serve as concentration barriers. Fractions B1 and B2 contained the largest cells with the lowest buoyant density and showed the highest expression levels for type X collagen (Col X), but only the B1 population expressed high levels of matrix metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly smaller and expressed little Col X, while cells in fraction B4 were of similar size to cells in the resting zone without significant Col X expression. The highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR-1), and Indian hedgehog (Ihh) expression were also found in the hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as expected from in situ hybridization data on PTHR-1 expression in fetal rodent or chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and PTHR-1 by more than 50% and the expression of Ihh nearly completely. In contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These findings confirm the existence of distinct differentiation stages within chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP fragments controlling the differentiation of proliferating to prehypertrophic chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic chondrocytes.