We investigated the effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) on cytosolic free calcium ([Ca2+]i) at a single cell level in both type-1 and type-2 cultured rat astrocytes using a calcium-sensitive fluorescent dye, fura-2. Type-1 astrocytes were relatively silent during the 20 min observation of baseline [Ca2+]i and PACAP38 did not alter [Ca2+]i in type-1 astrocytes at concentrations up to 1 microM. In contrast, type-2 astrocytes could be divided into three subtypes (silent type, randomly-firing type and oscillatory type) based on the observation of baseline [Ca2+]i. Of 166 type-2 astrocytes tested, 145 (87.4%) were the silent type (stable basal [Ca2+]i levels) and 13 (7.8%) were the randomly-firing type (random increases in [Ca2+]i). PACAP38 could stimulate [Ca2+]i in subpopulations of all three subtypes. In the silent type-2 astrocytes (4 experiments with 255 cells/experiment), 54.4 +/- 3.6% of the total number responded to PACAP38. The half maximal concentration (ED50) of PACAP38 was 2.89 +/- 1.89 nM. Removing Ca2+ from the superfusion media did not alter the PACAP38-induced increase of [Ca2+]i. Neither 1-30 microM of forskolin nor 1-10 mM of dibutyryl cyclic AMP increased [Ca2+]i in the same type-2 astrocytes which responded to PACAP38. These findings suggest that PACAP increases [Ca2+]i in type-2, not type-1, rat astrocytes by mobilizing Ca2+ from intracellular stores, and that this action is not linked to activation of the cyclic AMP second messenger system.