Longitudinal growth is a result of proliferation and differentiation of chondrocytes in the growth plate. Growth hormone (GH) stimulates longitudinal growth, and GH receptors have been shown on growth plate chondrocytes, but the effects of GH on chondrocytes of different cell layers are not clear. To study the effect of GH on chondrocyte activity, in situ biochemical techniques were used to measure enzyme activities, which are associated with cell differentiation (alkaline phosphatase [ALP]) and osteoclast activity (tartrate-resistant acid phosphatase [TRAP]), within single cells of the growth plate. Uptake of bromodeoxyuridine (BrdU) was used as a parameter for proliferative activity. In addition, glucose-6-phosphate dehydrogenase (G6PD) was measured since increased proliferation has been associated with increased G6PD activity. The role of GH was studied in a model of isolated GH deficiency (dwarf rat) and complete pituitary deficiency (hypophysectomized rat). Groups of GH-deficient dwarf rats were infused with recombinant human GH in either a continuous or a pulsatile manner, since the pattern of GH secretion is an important regulator of growth in the rat. After 7 days, G6PD activity in proliferative chondrocytes and TRAP activity in osteoclasts was increased, while ALP activity in hypertrophic chondrocytes was decreased. GH not only increased the number of chondrocytes that incorporated BrdU but also the total number of chondrocytes in the proliferative zone; therefore, its ratio, the labeling index (an indicator of proliferative rate), was not increased. The widths of the proliferative and hypertrophic zones were increased by both patterns of GH administration. The width of the resting zone was unaffected by continuous GH but decreased by pulsatile GH. ALP and TRAP activities were, respectively, higher and lower in hypophysectomized rats compared with the GH-deficient animals. Hypophysectomized rats had smaller growth plates than dwarf rats with a disproportionally wide resting zone, which, like BrdU uptake, was not affected by GH. GH treatment resulted in increased TRAP and decreased ALP activity. These results indicate that GH stimulates the commitment of chondrocytes within the resting/germinal layer to a proliferative phenotype (as opposed to stimulating the rate of chondrocyte proliferation) but only in the presence of other pituitary hormones. Furthermore, this study shows that enzyme activities within single chondrocytes and osteoclasts are GH-sensitive. The extent to which these effects are direct or mediated by systemic or local growth factors remains to be clarified.