We investigated the effects of tumor necrosis factor-α (TNFα), a cytokine produced as one aspect of inflammatory reactions, on the intracellular free calcium concentration in single pituitary cells using a calcium-sensitive fluorescent dye, indo-1, and a digital imaging fluorescence microscopic system. TNFα induced an increase in intracellular free calcium immediately after administration, reaching a peak after 30 sec and then returning to nearly the basal level after 120 sec in the pituitary cells. The cells responding to TNFα constitute about 15% of the pituitary cell population, and these cells never responded to the hypothalamic releasing hormones TRH, GnRH, GH-releasing hormone, or CRH. To define the role of calcium in TNFα-induced PRL release, dispersed pituitary cells were exposed to agents that modify TNFα-induced calcium mobilization. Such calcium channel blockers as cobalt and verapamil decreased basal and TNFα-induced PRL release. A low calcium medium also decreased TNFα-induced PRL release. These data suggest that intracellular calcium mobilization may be involved in the process of TNFα-induced PRL release. (Endocrinology128: 2785–2790,1991)