Postnatal sympathetic neurons (SNs) and chromaffin cells (CCs) derive from neural crest precursors. CCs can differentiate in vitro into SN-like cells after nerve growth factor (NGF) exposure. This study examines changes of intracellular Ca2+ homeostasis and dynamics of CCs under conditions that promote a neuronal phenotype. Spontaneous Ca2+ fluctuations, a frequent observation in early cultures of CCs, diminished after > 10 days in vitro in control cells and ceased in NGF-treated ones. At the same time, Ca2+ rises resulting from entry upon membrane depolarization, gradually increased both their size and peak d[Ca2+]i/dt, resembling those recorded in SNs. Concomitantly, caffeine-induced Ca2+ rises, resulting from Ca2+ release from intracellular stores, increased their size and their peak d[Ca2+]i/dt by > 1000%, and developed transient and sustained release components, similar to those of SNs. The transient component, linked to regenerative Ca2+ release, appeared after > 10 days of NGF treatment, suggesting a delayed steep enhancement of Ca2+-induced Ca2+ release (CICR). Immunostaining showed that proteins coded by the three known isoforms of ryanodine receptors (RyRs) are present in CCs, but that only RyR2 increased significantly after NGF treatment. Since the transient release component increased more steeply than RyR2 immunostaining, we suggest that the development of robust CICR requires both an increased expression of RyRs and more efficient functional coupling among them. NGF-induced transdifferentiation of chromaffin cells involves the enhancement of both voltage-gated Ca2+ influx and Ca2+ release from intracellular stores. These modifications are likely to complement the extensive morphological and functional reorganization required for the replacement of the endocrine phenotype with the neuronal one.