Abstract A detailed description is given of an immunohistochemical technique for the localization of serotonin in the central nervous system of the rat. In rabbits antibodies were raised against an antigen prepared by coupling serotonin to bovine serum albumin using formaldehyde as a coupling reagent. In the antisera two types of antibodies, one against the bovine serum albumin-serotonin complex and another type directed to bovine serum albumin were demonstrated by immunoelectrophoresis. The antibodies to serum albumin could be removed by affinity chromatography. Brains and spinal cords of rats were fixed by perfusion with ice-cold 4% formaldehyde in 0.1 m-sodium phosphate buffer (pH 7.3) and sectioned on a cryostat. The sections were stained by the indirect immunofluorescence technique. Immunofluorescence specific for serotonin was observed in cell bodies and fibre systems. The antisera could be diluted up to 1:1024 provided a prolonged incubation at 4°C was employed. The specificity of the antisera was examined by inhibition tests. From these tests it was estimated that in the central nervous system of the rat there might be a cross-reactivity of approximately 2% to 5-methoxytryptamine and dopamine and less than 1% to noradrenaline and adrenaline under the conditions which were used. Thus the procedure appears to be a specific and sensitive technique for the localization of serotonin in the central nervous system; it has the advantage that adjacent sections can be examined by the immunofluorescence method using antisera to a variety of antigens. The application of this technique is some parts of the brain and spinal cord of the rat is demonstrated.