Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.