Prolactin secretion is stimulated markedly in fish exposed to water of pH 4. This phenomenon was used to study the control of prolactin secretion. Activation occurs irrespective of changes in plasma osmolarity or plasma sodium and calcium concentrations. After acute acidification of the water, which leads to a substantial fall in plasma osmolarity and plasma electrolyte levels, the activation of the prolactin cells is less marked than after gradual acidification of the water, when plasma osmolarity, plasma sodium, and plasma total and ionic calcium levels are not noticeably affected. When fish bearing an implanted rostral pars distalis of the pituitary gland are exposed to water of pH 4, both the in situ prolactin cells and the prolactin cells of the implant become activated only when the drop in water pH is acute and followed by a reduction in plasma osmolarity and electrolyte levels. When the rate of reduction of the pH is slow and not changing plasma osmolarity or sodium and calcium levels, the in situ prolactin cells are stimulated, but not those of the implants. We conclude that the activation of the prolactin cells in situ in fish in acid water is not mediated by reductions in plasma osmolarity, plasma sodium, total calcium, or ionic calcium, but by hypothalamic mechanisms. The drop in plasma osmolarity and electrolytes probably reflects excessive osmoregulatory stress and this may hamper instead of stimulate the response of the prolactin cells to osmoregulatory disturbance. The physiological significance of the in vitro activation of prolactin cells by reduced ambient osmolarity is unclear.