The nitric oxide (NO) synthase inhibitor N-nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45°C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC₅₀ of 0.0471 ± 0.0007 µM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca²⁺ dependent, we measured [Ca²⁺]_i after treating cells with L-NNA. L-NNA at 100 µM significantly decreased resting [Ca²⁺]_i. Likewise, treatment with 1 µM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca²⁺]_i and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca²⁺]_i and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca²⁺]_i and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.