The goal of this study was to examine the expression and regulation of aquaporin2 (AQP2), a tonicity-sensitive water channel in nucleus pulposus cells of the intervertebral disc. We found that AQP2 protein was expressed in vivo in both rat and human discs. We determined whether AQP2 promoter expression was regulated by osmolarity in a tonicity enhancer binding protein (TonEBP)-dependent manner. When TonEBP was suppressed under hypertonic conditions or overexpressed under isotonic conditions, AQP2 promoter activity was correspondingly inhibited or induced. The role of TonEBP in controlling AQP2 expression was confirmed using mouse embryonic fibroblasts (MEFs) derived from TonEBP-null mice. We studied whether calcium in addition to osmolarity played a role in regulation of AQP2 in nucleus pulposus cells. We also determined whether both TonEBP and calcineurin–nuclear factor of activated T cells (NFAT) signaling contributed to ionomycin, a calcium ionophore, mediated induction of AQP2. Co-transfection of AQP2 reporter with calcineurin (CnA/B) and/or NFAT1–4 vectors suggested that this pathway did not control AQP2 promoter activity in nucleus pulposus cells. These findings were also validated using MEFs from TonEBP, fibroblasts from CnAα- and CnAβ-null mice, and mutant TonE reporter constructs. Results of these studies suggest that, in nucleus pulposus cells, osmotic pressure and calcium modulate AQP2 expression through TonEBP and are independent of the calcineurin–NFAT pathway. Because calcium flux reflects a change in applied stress, the possibility exists that NFAT5/TonEBP modulate not just water balance in the disc but also accommodate applied biomechanical forces.