This review discusses the regulation of growth plate chondrocytes by vitamin D₃. Over the past ten years, our understanding of how two vitamin D metabolites, 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃, exert their effects on endochondral ossification has undergone considerable advances through the use of cell biology and signal transduction methodologies. These studies have shown that each metabolite affects a primary target cell within the endochondral developmental lineage. 1α,25-(OH)₂D₃ affects primarily growth zone cells, and 24R,25-(OH)₂D₃ affects primarily resting zone cells. In addition, 24R,25-(OH)₂D₃ initiates a differentiation cascade that results in down-regulation of responsiveness to 24R,25-(OH)₂D₃ and up-regulation of responsiveness to 1α,25-(OH)₂D₃. 1α,25-(OH)₂D₃ regulates growth zone chondrocytes both through the nuclear vitamin D receptor, and through a membrane-associated receptor that mediates its effects via a protein kinase C (PKC) signal transduction pathway. PKCα is increased via a phosphatidylinositol-specific phospholipase C (PLC)-dependent mechanism, as well as through the stimulation of phospholipase A₂ (PLA₂) activity. Arachidonic acid and its downstream metabolite prostaglandin E₂ (PGE₂) also modulate cell response to 1α,25-(OH)₂D₃. In contrast, 24R,25-(OH)₂D₃ exerts its effects on resting zone cells through a separate, membrane-associated receptor that also involves PKC pathways. PKCα is increased via a phospholipase D (PLD)-mediated mechanism, as well as through inhibition of the PLA₂ pathway. The target-cell-specific effects of each metabolite are also seen in the regulation of matrix vesicles by vitamin D₃. However, the PKC isoform involved is PKCζ, and its activity is inhibited, providing a mechanism for differential autocrine regulation of the cell and events in the matrix by these two vitamin D₃ metabolites.