The combination of Ca²⁺ influx during neurotransmission and low cytosolic Ca²⁺ buffering contributes to the preferential vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS). This study investigated the relationship among Ca²⁺ accumulation in intracellular compartments, mitochondrial abnormalities, and protein aggregation in a model of familial ALS (fALS1). Human SOD1, wild type (SOD1^WT) or with the ALS-causing mutation G93A (SOD1^G93A), was expressed in motor neurons of dissociated murine spinal cord–dorsal root ganglia (DRG) cultures. Elevation of mitochondrial Ca²⁺ ([Ca²⁺]_m), decreased mitochondrial membrane potential (Δψ) and rounding of mitochondria occurred early, followed by increased endoplasmic reticular Ca²⁺ ([Ca²⁺]_ER), elevated cytosolic Ca²⁺ ([Ca²⁺]_c), and subsequent appearance of SOD1^G93A inclusions (a consequence of protein aggregation). [Ca²⁺]_c was elevated to a greater extent in neurons with inclusions than in those with diffusely distributed SOD1^G93A and promoted aggregation of mutant protein, not vice versa: both [Ca²⁺]_c and the percentage of neurons with SOD1^G93A inclusions were reduced by co-expressing the cytosolic Ca²⁺-buffering protein, calbindin D-28K; treatment with the heat shock protein inducer, geldanamycin, prevented inclusions but not the increase in [Ca²⁺]_c, [Ca²⁺]_m or loss of Δψ, and inhibiting proteasome activity with epoxomicin, known to promote aggregation of disease-causing mutant proteins including SOD1^G93A, had no effect on Ca²⁺ levels. Both expression of SOD1^G93A and epoxomicin-induced inhibition of proteasome activity caused mitochondrial rounding, independent of Ca²⁺ dysregulation and reduced Δψ. That geldanamycin prevented inclusions and mitochondrial rounding, but not Ca²⁺ dysregulation or loss of Δψ indicates that chaperone-based therapies to prevent protein aggregation may require co-therapy to address these other underlying mechanisms of toxicity.