We have recently developed a model system for testing the necessity, appropriateness and sufficiency of individual components of the respiratory central pattern generator (CPG) in Lymnaea stagnalis. In order to examine the intrinsic and network properties of the three CPG interneurons (R.Pe.D1, V.D4 and Ip.3.I), these cells were isolated and cultured in vitro. All of these cells exhibited extensive neurite outgrowth within 18-24 h of plating in conditioned medium. These isolated neurons maintained their intrinsic properties in culture and also formed specific synapses with each other similar to those observed in vivo. However, only when all three interneurons were plated together was it possible to initiate the alternating rhythm characteristic of the respiratory activity observed in semi-intact or isolated brain preparations.