Abstract The subcellular distribution of serotonin (5-hydroxytryptamine; 5-HT) in the lamprey ( Ichtyomyzon unicuspis, Lampetra fluviatilis) spinal cord was investigated by using ultracentrifugation on continuous density gradients combined with an electron microscopic analysis of the gradients and of immunostained tissue. Endogenous 5-HT was analyzed by high-performance liquid chromatography with electrochemical detection. After differential centrifugation, the highest levels of 5-HT were found in the particulate fractions. After ultracentrifugation of lysed synaptosomal fractions on continouus sucrose gradients and the subsequent sedimentation of the individual fractions, 5-HT showed a biphasic distribution in the gradient. The two peaks corresponded to 0.30–0.40 M and 0.85–1.05 M sucrose. Electron microoscopy of intact tissue showed that some of the boutons were strongly immunoreactive to 5-HT with dense precipitates over large granular vesicles. The area around these large vesicles, however, also showed reaction product. Large granular vesicles could be clearly distinguished in the immunostained axonal varicosities. In tissue not processed for 5-HT immunoreactivity it was seen that the varicosities contained not only large dense-cored vesicles, but also small agranular vesicles. An electron microscopical analysis of the subcellular fractions revealed that the fraction corresponding to the “light” 5-HT peak contained numerous vesicular structures, which in most cases were electron lucent. In the “heavy” fractions, nerve ending particles containing vesicles of various sizes were observed. The results suggest that 5-HT in the lamprey spinal cord may be distributed in more than one subcellular compartment wwhich, apart from the cytosol, possibly corresponds to small and large synaptic vesicles.