OBJECTIVE To understand the molecular basis of early orthodontic tooth movement by looking at the expression of KI-67, runt-related transcription factor 2 (Runx2), and tumor necrosis factor ligand superfamily member 11 (RANKL) proteins. METHODS We employed a rat model of early orthodontic tooth movement using a split-mouth design (where contralateral side serves as a control) and performed immunohistochemical staining to map the spatial expression patterns of three proteins at 3 and 24 hours after appliance insertion. RESULTS We observed increased expression of KI-67, a proliferation marker, and RANKL, a molecule associated with osteoclastic differentiation, in the compression sites of the periodontal ligament subjected to 3 hours of force. In contrast, there was increased expression of KI-67 and Runx2, a marker of osteoblast precursors, in tension areas after 24 hours of force. Decreased KI-67 expression in the mesial and distal regions of the periodontal ligament was observed at the midpoint of the tooth root. CONCLUSIONS The early RANKL expression indicates that at this early stage cells are involved in osteoclast precursor signaling. Also, decreased KI-67 expression found near the midpoint of the tooth root is believed to represent the center of rotation, providing a molecular means of visualizing mechanical loading patterns.