The giant cholinergic neuron R2 of Aplysia was cultured in combination with identified neurons L11 and R15 and members of a group of left upper quadrant (LUQ) cells L2 to L6 from the abdominal ganglion. All of these neurons receive cholinergic input from other cells in vivo, but not from R2. In vitro, R2 reliably formed unidirectional chemical connections with these cells. Single action potentials in R2 produced a dual fast and slow inhibitory response in LUQ cells (L2 to L6), a dual fast inhibitory-slow excitatory response in L11, and a slow inhibitory response in R15. The connections formed on LUQ cells were characteristic of their cholinergic input, but the R2-L11 and the R2-R15 connections also had noncholinergic properties. Thus, unlike L10 which forms connections only with its normal targets in vitro, R2 forms strong chemical connections with other neurons which are not found in vivo. The properties of the R2 connections also suggest that it may release another neurotransmitter besides acetylcholine.