Electrophysiological analysis of cultured neurons provides a potential approach toward understanding the physiological defects that may contribute to abnormal behavior exhibited by mutants of the fruit fly Drosophila. However, its application has been restricted by an inability to identify a particular functional or anatomical subpopulation of neurons from the CNS. To study neurons composing the CNS mushroom body proposed as a center for insect olfactory learning, we utilized a Drosophila enhancer detector line that expresses a lacZ reporter gene in these neurons and identified them in acutely dissociated larval CNS cultures by vital fluorescent staining. The patch-clamp analysis suggests that whole-cell voltage-activated K+ currents can be classified into two types in identified mushroom body neurons. Type 1 current comprises a TEA-sensitive slowly inactivating current and noninactivating component while type 2 current contains a 4-AP-sensitive transient A-current and a noninactivating component. Application of cAMP analogs induced distinct modulation of type 1 and type 2 currents. Our results demonstrate that the expression of the lacZ gene and the subsequent staining do not significantly alter the different types of K+ currents. This initial characterization provides a basis for further analysis of mutations that impair learning and memory resulting from an abnormal cAMP cascade preferentially expressed in the mushroom body.