The effects of parathyroid hormone on the cytoplasmic Ca2+ concentration of canine renal proximal tubule cells grown in primary culture were determined using the fluorescent Ca2+ indicator quin 2. The cultured cells exhibited responses to hormones, enzyme activities, transport functions, and morphology characteristic of the proximal convoluted tubule. Parathyroid hormone stimulated an immediate rise in cytoplasmic Ca2+, both in suspended cells and cells studied as a monolayer on Nuclepore filters. The rise in cytoplasmic Ca2+ induced by the hormone was sustained for 15-30 min, was dose dependent, and was not mimicked by cyclic AMP. Removing Ca2+ from the extracellular media markedly decreased cytoplasmic Ca2+ and abolished the effects of parathyroid hormone on cytosolic Ca2+. 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate blocked the effects of the hormone on cytosolic Ca2+, but mitochondrial uncouplers failed to inhibit the effects of the hormone to increase cytoplasmic Ca2+. These studies support a role of Ca2+ in the activation of proximal renal tubule cells by parathyroid hormone.