We have investigated alterations in Ca²⁺ entry mechanisms following differentiation of SH-SY5Y cells with 9-cis retinoic acid (9cRA). Fura-2 fluorescence measurements were used to monitor changes in [Ca²⁺]_i and divalent cation entry (Mn²⁺ quench). Fluorescence from fura-2 loaded cells was monitored using an LS50B fluorimeter (Perkin Elmer) with excitation and emission wavelengths of 340 and 510nm respectively to monitor changes in [Ca²⁺]_i, and 360 and 510nm respectively to monitor Mn²⁺ quench (Bennett et al., 1998). The muscarinic agonist methacholine stimulated a [Ca²⁺]_i rise in both the presence and absence of Ca²⁺ . Dose-response relationships revealed that in undifferentiated cells, peak [Ca²⁺]_i responses were significantly increased in the presence of Ca²⁺ above those in its absence, whereas in differentiated cells, responses were similar regardless. Ca²⁺ entry was directly investigated using a Ca²⁺ addback technique (e.g. Moneer & Taylor, 2002). Thapsigargin stimulated significant Ca²⁺ entry in undifferentiated but not differentiated cells, whilst methacholine elicited significant Ca²⁺ entry in both. Thapsigargin stimulated an increase in Mn²⁺ quench in undifferentiated but not differentiated cells. Methacholine also stimulated an increased rate of quench in undifferentiated but not differentiated cells, suggesting that methacholine-stimulated Ca²⁺ entry in undifferentiated cells is Mn²⁺ permeable, whereas that in differentiated cells is Mn²⁺ impermeable. Also, methacholine stimulated a significantly higher Mn²⁺ entry rate in undifferentiated cells than thapsigargin. This, plus the finding of a non-CCE, Mn²⁺ impermeable pathway in differentiated cells, implied that methacholine may stimulate more than one entry pathway. Mn²⁺ entry rates were therefore measured in undifferentiated cells after the sequential addition of thapsigargin and methacholine. Separate experiments monitored these rates with this order of addition reversed. Thapsigargin stimulated increased Mn²⁺ entry and subsequent methacholine addition stimulated a further increase. When methacholine was added first, an increased rate was observed but no further increase was detected upon thapsigargin addition. Hence, methacholine may stimulate a divalent cation entry pathway in addition to CCE. In summary, we have shown that CCE is apparently down-regulated upon 9cRA-induced differentiation of SH-SY5Y cells, and this appears to be accompanied by the up-regulation of another, as yet unidentified methacholine-stimulated entry pathway.