There is evidence that the signal transduction array responsible for the secretion of water in evaporative cooling by the submaxillary gland of the rat is subject to heat acclimatory responses. The objectives of the present study were 1) to examine whether heat acclimation affects intracellular Ca²⁺ mobilization and, in turn, submaxillary glandular responsiveness; 2) to assess whether the acclimatory responses differ from those evoked on heat stress (HS). Experiments were conducted on submaxillary glands of rats acclimated at 34°C for 0, 2 [short-term heat acclimation (STHA)], and 30 [long-term heat acclimation (LTHA)] days. The resting cytosolic calcium concentration ([Ca²⁺]_c) and the carbamylcholine-evoked calcium signal ([Ca²⁺]_s) of dispersed glandular cells were measured using the fluorescent dye fura 2 AM. Inositol-1,4,5-trisphosphate (IP₃)-sensitive endoplasmic reticulum Ca²⁺ stores were determined in permeabilized cells using fura 2 potassium salt. STHA resulted in a drop in both [Ca²⁺]_s and IP₃-sensitive Ca²⁺ stores. On LTHA, the [Ca²⁺]_s amplitude reverted to the preacclimation value, whereas the IP₃-sensitive Ca²⁺ stores remained low. The drop in [Ca²⁺]_s on STHA is in accord with the decreased glandular output (measured by ⁸⁶Rb efflux) observed during this acclimation phase. However, after LTHA the enhanced glandular output despite reduced [Ca²⁺]_s levels suggests an increased efficiency of cellular secretory mechanisms in that group. Collectively, the alterations in [Ca²⁺]_s support our biphasic acclimation model (Horowitz M, Kaspler P, Marmari Y, and Oron Y. J Appl Physiol 80: 77-85, 1996.). In nonacclimated glands, HS caused an elevation in [Ca²⁺]_s coincidentally with a decrease in the IP₃ Ca²⁺ stores. In contrast, [Ca²⁺]_s in both STHA and LTHA glands was not affected by HS, despite a marked increase in the IP₃-sensitive Ca²⁺ stores in the LTHA glands. The opposing responses to HS and heat acclimation in calcium signaling and stores confirm the specificity of each process.