The rectal gland of Squalus acanthias is critically involved in the homeostasis of NaCl and water metabolism and hence in overall osmoregulation. In the present study, we have examined the acute responses of rectal gland slices and in vitro perfused rectal gland tubule (RGT) cells to the exposure to dilute and hypertonic peritubule solutions. Five series were performed. (i) With changes in osmolality, Western blots to monitor tyrosine, threonine and serine phosphorylation in rectal gland slices did not reveal clear–cut changes in phosphorylation patterns. All other series were performed in in vitro perfused RGT. (ii) Relative cell volume was estimated by fura–2 fluorescence using the emission at the isosbestic excitation wavelength of 360 nm. Hypotonic solution (–100 mmol/l NaCl) reduced fura–2 fluorescence by 16% and hypertonic solution (+100 mmol/l NaCl) had the opposite effect (+12%). (iii) Transepithelial resistance was increased markedly by hypotonic solution, probably by cell swelling, and the opposite was seen with hypertonic solutions. (iv) Whole–cell patch clamp experiments indicated that hypotonic solution hyperpolarized the cells, and increased membrane conductance and membrane capacitance. The latter two changes correlated significantly with each other. Hypertonic solution had the opposite effect. (v) Measurements of the fura–2 fluorescence ratio (340/380 nm) revealed that hypotonic solution (–NaCl) increased cytosolic Ca<sup>2+</sup> activtiy ([Ca<sup>2+</sup>]<sub>i</sub>). Hypertonic solution had no detectable effect on [Ca<sup>2+</sup>]<sub>i</sub>. These data indicate that RGT cells are swollen by removal of NaCl from the bath solution. This causes an increase in [Ca<sup>2+</sup>]<sub>i</sub> and a predominant increase in K<sup>+</sup> conductance and hyperpolarization. Urea apparently permeates these cells quite well and its addition (+U) or its removal (–U) had only moderate osmotic effects. The removal of urea and replacement by mannitol produced effects similar to those seen with hypertonic NaCl solution.