Of the many factors that regulate linear growth, insulin-like growth factor-I (IGF-I) has a central role in epiphyseal chondrocyte development. Whether IGF-I is solely of systemic or also of local origin is uncertain, as is how other growth factors interact with IGF-I at the growth plate. We studied the proliferative effects of IGF-I on juvenile bovine epiphyseal chondrocytes fractionated by density gradient centrifugation. Cell density correlated with size, glycogen content and gene expression patterns. There was a gradient of response to IGF-I, with the greatest proliferative response in high density cells corresponding to the reserve zone, as measured by ³H-thymidine uptake. Low-density (hypertrophic zone) cells proliferated only when exposed to IGF-I and basic fibroblast growth factor (bFGF). The gradient of IGF-I response correlated with ¹²⁵I-IGF-I binding as determined by Scatchard analysis: IGF-I receptor number was ten-fold greater in reserve zone cells than in hypertrophic cells. When exposed to bFGF for 24 hours, IGF-I binding in hypertrophic cells increased threefold. In contrast, no specific binding of growth hormone (GH) was demonstrated in juvenile bovine chondrocytes. GH produced neither signal transducer and activator of transcription (STAT) phosphorylation, nor increased proliferation, nor increased IGF-I mRNA levels in any chondrocyte fraction. IGF-I mRNA levels were extremely low at 800-1100 copies/µg 18S RNA in bovine chondrocytes. These results suggest that the major regulator of chondrocyte proliferation is systemic IGF-I; FGFs may influence the actions of IGF-I at the growth plate by altering its receptor number in chondrocytes.