Histochemical and indirect immunocytochemical techniques were used to search for neuroactive substances and transmitter candidates in identified sensory neurons of two types of cuticular mechanoreceptors in the spider Cupiennius salei Keys.: (1) in lyriform slit-sense organ VS-3 (comprising 7-8 cuticular slits each innervated by 2 bipolar neurons), and (2) in tactile hairs (each supplied by 3 bipolar sensory cells). All neurons are mechanosensitive. A polyclonal antibody against choline acetyltransferase (ChAT) strongly labeled all cell bodies and afferent fibers of both mechanoreceptor types. Western blot analysis using the same antibody against samples of spider sensory hypodermis and against samples from the central nervous system demonstrated a clear band at 65 kDa, corresponding to the molecular mass of ChAT in insects. Moreover, staining for acetylcholine esterase (AChE) revealed AChE activity in one neuron of each mechanoreceptor type. Incubation with a polyclonal antibody against histamine clearly labeled one neuron in each set of sensilla, whereas activity in the remaining one or two cells was near background. All mechanoreceptor preparations treated with a polyclonal antiserum against serotonin tested negative, whereas sections through the central nervous system of the same spiders were clearly labeled for serotonin. The presence of ChAT-like immunoreactivity and AChE implicates acetylcholine as a transmitter candidate in the two mechanoreceptive organs. We assume that histamine serves as a mechanosensory co-transmitter in the central nervous system and may also act at peripheral synapses that exist in these sensilla.