Nineteen ortho-substituted PCBs are chiral and found enantioselectively enriched in ecosystems. Their differential actions on biological targets are not understood. PCB 95 (2,2',3,5',6-pentachlorobiphenyl), a chiral PCB of current environmental relevance, is among the most potent toward modifying ryanodine receptors (RyR) function and Ca2+ signaling. PCB 95 enantiomers are separated and assigned aR- and aS-PCB 95 using three chiral-column HPLC and circular dichroism spectroscopy. Studies of RyR1-enriched microsomes show aR-PCB 95 with >4× greater potency (EC50 = 0.20 ± 0.05 μM), ∼ 1.3× higher efficacy (Bmax = 3.74 ± 0.07 μM) in [3H]Ryanodine-binding and >3× greater rates (R = 7.72 ± 0.31 nmol/sec/mg) of Ca2+ efflux compared with aS-PCB 95, whereas racemate has intermediate activity. aR-PCB 95 has modest selectivity for RyR2, and lower potency than racemate toward the RyR isoform mixture in brain membranes. Chronic exposure of hippocampal neuronal networks to nanomolar PCB 95 during a critical developmental period shows divergent influences on synchronous Ca2+ oscillation (SCO): rac-PCB 95 increasing and aR-PCB 95 decreasing SCO frequency at 50 nM, although the latter's effects are nonmonotonic at higher concentration. aS-PCB95 shows the greatest influence on inhibiting responses to 20 Hz electrical pulse trains. Considering persistence of PCB 95 in the environment, stereoselectivity toward RyRs and developing neuronal networks may clarify health risks associated with enantioisomeric enrichment of PCBs.