Proteoglycans have been implicated in neuronal pathfinding during development, yet related second messenger and signaling systems are unknown. We have used the calcium indicator fura-2/AM to monitor cytoplasmic calcium ion concentration ([Ca²⁺]_i) in chick dorsal root ganglion (DRG) neuronal growth cones elongating on laminin during contact with chondroitin sulfate proteoglycan (CSPG): (1) to determine whether there is a change in [Ca²⁺]_i in neurons that contact CSPG, and (2) to determine whether changes in [Ca²⁺]_i are necessary for inhibition of growth cone migration. The majority of DRG neurons responded to CSPG contact with a transient rise in [Ca²⁺]_i (mean Δ[Ca²⁺]_i above resting level was 554 ± 109 nM; P < 0.0001). The effect of CSPG contact was concentration dependent and required the carbohydrate moiety of CSPG. Addition of soluble CSPG did not elevate [Ca²⁺]. Treatment with reagents that blocked plasma membrane calcium channels, or that perturbed intracellular Ca²⁺ stores, indicated that extracellular Ca²⁺ was the major source of the [Ca²⁺]_i elevation, and that Ca²⁺ entry occurred through non-voltage-gated calcium channels. Although general Ca²⁺ channel blockers abolished the CSPG-induced [Ca²⁺]_i rise, they did not abolish growth cone avoidance of surface-bound CSPG in these assays. We conclude: (1) that DRG neurons elevate [Ca²⁺]_i in response to CSPG contact to levels that can modify cytoskeletal mechanisms of growth cone migration, and (2) that avoidance of substratum-bound CSPG may not be dependent upon elevated [Ca²⁺]_i.