This study examined the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and related compounds on the uptake of intracellular calcium ([Ca2 +]i) in primary cultures of rat hippocampal neuronal cells. [Ca2 +]i levels were detected and quantified by interactive laser cytometry with microscopic image analysis. Cells were noninvasively labeled with fluo-3/AM and all experiments were conducted on cultured rat hippocampal neurons 14 days in culture. Treatment of cell cultures with 2,3,7,8-TCDD (10–100 nM) resulted in a rapid concentration-dependent increase in [Ca2 +]i associated with a decrease in mitochondrial membrane potential and activation of α-protein kinase C (α-PKC). In contrast, 1,2,3,4-TCDD, a weak Ah receptor agonist, had no effect on [Ca2 +]i at concentrations as high as 10 μM and similar results were also observed for 2,2′,5,5′-tetrachlorobiphenyl. Maximal [Ca2 +]i was observed within 30 s after addition of 2,3,7,8-TCDD and remained elevated (at higher concentrations) above resting levels for the duration of the experiment. This rapid increase in [Ca2 +]i was blocked by addition of EDTA (2 mM) to the external medium or by pretreatment of the cells with the calcium channel antagonist nifedipine (10 μM). However, pretreatment of the cells with 100 μM cycloheximide failed to block calcium uptake in neuronal cells. These data indicate that rat hippocampal neuronal cells are responsive to 2,3,7,8-TCDD; however, the mechanism is not associated with altered gene transcription and may involve cellular membrane targets.