Ca²⁺ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these Ca²⁺‐dependent effects is nuclear. Since cytoskeletal proteins are rapidly degraded by Ca²⁺‐dependent proteinases (calpains) in vitro and in vivo, we investigated whether Ca²⁺‐induced pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal‐like neurons were cultured and the ability of the membrane‐permeable calpain inhibitors ethyl(+)‐(2S,3S)‐3‐[(S)‐methyl‐1‐(3‐methylbutylcarbamoyl)‐butylcarbamoyl]‐2‐ oxiranecarboxylate (EST) and carbobenzoxyl‐valyl‐phenylalanyl‐H (MDL 28170) to block the Ca²⁺ ionophore A23187‐induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a significant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST (5 or 20 m̈M) to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 (20 m̈M) showed the same effect as EST in preventing ionophore‐induced pruning of dendrites and axons at 100 and 300 nM concentrations, respectively, of A23187. EST (20 m̈M) did not block the A23187‐induced rise of [Ca²⁺]_i as measured with fura‐2. These results suggest that calpains play a role in Ca²⁺‐induced pruning of neurites in isolated hippocampal pyramidal neurons. © 1994 Wiley‐Liss, Inc.