We have developed a primary cell culture system of antennal mechanoreceptor neurons from early-stage pupal sphinx moth Manduca sexta. Dissociated neurons from the moth antennae differentiated, grew and survived for several weeks in a conditioned culture medium. Bipolar neurons with soma diameters of 10-25 microns from the basal portion of the antennae could be positively identified as mechanoreceptor neurons, presumably derived from Johnston's organ, using a monoclonal antibody that recognizes neurofilaments in these neurons. The immunoreactivity was clear and specific from the first day after dissociation and became stronger during several days in culture. These neurons appeared healthy and showed normal whole-cell properties only a few days after plating. We found numerous mechanosensitive ion channels responding to both negative and positive pressures on the somata and neurites of differentiated neurons. This new culture system provides access to mechanoreceptor neurons that has never been possible before, allowing the use of both mechanical and electrical stimuli on neurons that are free from the accessory structures surrounding them in intact preparations.