A novel insulin-like immunoreactivity, adrenergic receptors (ARs) and voltage-dependent Ca[superscript]2+ channels (VDCCs) were characterized in a clonal [beta]-cell line, RINm5F, in a passage- and morphology-dependent manner. The cells exhibited two morphological features; the differentiated cells (DCs) with long neuron-like processes and the undifferentiated cells (UCs) without the processes;As morphology of the cells changed from DCs to UCs, amount of secreted insulin gradually decreased; however, insulin reacted to antiserum vs. bovine insulin (ABI) gradually increased from barely detectable amounts. Immunoblotting and immunocytochemistry showed that antiserum vs. rat insulin reacted with insulin from both cell types. ABI did not react with insulin from DCs but from UCs;cAMP production and insulin release by epinephrine were decreased in DCs but increased in UCs. In DCs, epinephrine decreased KCl-induced insulin release but RX 821002 abolished the epinephrine-induced inhibition. Prazosin and propranolol failed to alter epinephrine's effect. In UCs, however, epinephrine enhanced KCl's action. RX 821002 further potentiated the effect of epinephrine on KCl's action. Propranolol abolished the effect of epinephrine on KCl's action but prazosin did not;Cholera toxin equally increased cAMP production in DCs and UCs. Effects of forskolin and IBMX on cAMP production were greater in UCs than in DCs. Activity of protein kinase A was not significantly different between DCs and UCs;Effects of Bay K 8644 and nimodipine on Ca[superscript]2+ currents, (Ca[superscript]2+) [subscript] i and insulin release were greater in DCs than in UCs. Nimodipine (1 [mu]M) failed to inhibit control Ca[superscript]2+ currents at the holding potentials of -80 mV but inhibited them by ~70% at O mV. The current-voltage relationship for activation and inactivation of Ca[superscript]2+ currents shifted to more negative potentials in DCs than in UCs;In summary, these findings suggested that a novel insulin-like immunoreactivity is present in UCs, which is not due to rat insulin I nor II. DCs have greater activity of [alpha][subscript]2-, but lesser activity of [beta]-ARs than UCs. Increased activity of [beta]-ARs in UCs is due partly to increased activity of adenylyl cyclase. DCs have greater activity of L-type VDCCs than UCs. The voltage-dependence of L-type VDCCs was different between DCs and UCs.