SH-SY5Y neuroblastoma cells undergo retinoid-induced morphological and biochemical differentiation (Pahlman et al., 1984). Understanding this differentiation process will form the basis for development of novel retinoid-based chemotherapeutic regimes for the treatment of neuroblastoma disease. Fura-2 fluorescence was measured in single SH-SY5Y cells, in order to characterise a range of Ca²⁺ signalling machinery in both undifferentiated and 9 cis-retinoic acid (9cRA)-differentiated cells. Cells grown on coverslips were loaded with fura-2 AM and mounted on the stage of a Nikon Diaphot epi-fluorescence microscope. Fura-2 fluorescence was excited alternately at 340 and 380nm. Emitted fluorescence was detected at 510nm and digitised by a charge-coupled device camera (Brown et al., 2002). Cells were depolarised with high (55mM) [K⁺]_e.The relative proportion of responsive cells differed: 63% of 9cRA-differentiated cells responded with a rise in [Ca²⁺]_i of ≥ 10nM, compared to only 45% of undifferentiated cells (n=118 and 92 respectively). Maximally effective concentrations of verapamil (L-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (ω-CgTx; N-type blocker) were separately found to partially and reversibly inhibit the Ca²⁺ response. In both cases the degree of inhibition was affected by differentiation; in differentiated cells ω-CgTx-induced inhibition was reduced (63 to 47%; n≥50) whilst verapamil-induced inhibition was increased (78 to 85%; n≥50). Thus, changes in either expression or function of L- or N-type voltage-operated Ca²⁺ channels could contribute to changes in excitability that accompany differentiation. A small proportion (≤ 3%) of undifferentiated cells produced a Ca²⁺ signal in response to the ryanodine receptor (RyR) agonist caffeine (30mM), which was blocked by pre-incubation with ryanodine (10µM). Comparable caffeine responses were observed in 9cRA-differentiated cells. In response to methacholine it was found, using a dual-application protocol, that the second Ca²⁺ signal was downregulated compared to the first. In undifferentiated cells, the presence of ryanodine was found to protect against this downregulation, whilst in 9cRAdifferentiated cells ryanodine had no protective effect. These data suggest that, in differentiated cells, RyR expression may be reduced, or that RyRs are present but non-functional in the response to methacholine. We have shown that both plasma membranelocalised and ER-localised components of the Ca²⁺-signalling ‘toolkit’ can alter in a functional manner following differentiation of SH-SY5Y cells.