It is reported here that male-male courtship behavior is evoked instantaneously in the fruit fly Drosophila by conditional disruption of synaptic transmission. A temperature-sensitive allele of the Drosophila dynamin gene shibire (shi(ts1)) was expressed by using the GAL4/UAS system to disrupt synaptic transmission from GAL4-positive neurons in a temperature-dependent manner. An enhancer-trap GAL4 line C309 directing shi(ts1) expression in central and peripheral neurons (C309/UAS-shi(ts1)) initiated stereotypical precopulatory behavior toward other mature males immediately after a temperature shift from the permissive to restrictive temperature. At the restrictive temperature, C309/UAS-shi(ts1) males formed "courtship chains" and exhibited abnormally high levels of head-to-head interactions. The temperature-induced male-male courtship is attributable not to an increase in sexual attractiveness but to an increase in sexual activity of C309/UAS-shi(ts1) males. Interestingly, the temperature-induced increase in sexual activity is specific toward male partners, because C309/UAS-shi(ts1) males courted receptive virgin females less vigorously and copulated less efficiently after shifted to the restrictive temperature. Among the GAL4-positive neurons in C309, conditional disruption of certain cholinergic neurons but not the mushroom body intrinsic neurons plays a critical role in the induction of male-male courtship. These neurons may be involved in inhibitory systems that normally suppress aberrant male-male courtship. The presented strategy that can induce behavioral abnormalities by disrupting synaptic transmission in an acute and noninvasive manner will allow further exploration as to how distinct neuronal groups control sexual orientation and other aspects of reproductive behavior in Drosophila.