Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca²⁺]_i) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca²⁺]_i changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca²⁺ channels (VGCC). ATP (10 μM) caused increased [Ca²⁺]_i transients (226.6±16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17β-estradiol (100 nM) inhibited ATP-induced [Ca²⁺]_i (164±14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17α-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 μM) and specific estrogen receptor antagonist ICI 182780 (1 μM) blocked the estradiol inhibition of ATP-induced [Ca²⁺]_i transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca²⁺]_i, suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca²⁺]_i transients was mediated by estrogen action on VGCC. Nifedipine (10 μM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca²⁺]_i transients. N- and P-type VGCC antagonists ω-conotoxin GVIA (1 μM) and ω-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca²⁺]_i transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca²⁺]_i flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca²⁺]_i transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system.