The nicotinic acetylcholine receptors (nAChRs) and the 5-HT3 serotonin receptor subtype belong to a superfamily of neurotransmitter-gated ion channels involved in fast synaptic communication throughout the nervous system. Their trafficking to the neuron plasmalemma, as well as their targeting to specific subcellular compartments, is critical for understanding their physiological role. In order to investigate the cellular distribution of these receptors, we tagged the N-termini of alpha3beta4-nAChR subunits and the 5-HT3AR subunit with cyan and yellow fluorescent proteins (CFP, YFP). The fusion subunits were coexpressed in human embryonic kidney (HEK-293) cells, where they assemble into functional receptor channels, as well as in primary cultures of hippocampal neurons. Fluorescence microscopy of living cells revealed that the heteropentameric alpha3CFP-beta4 and YFP-alpha3beta4 receptors are mainly distributed in the endoplasmic reticulum, while the homopentameric YFP-5-HT3A receptor was localized both to the plasma membrane and within intracellular compartments. Moreover, the YFP-5-HT3A receptor was found to be targeted to the micropodia in HEK-293 cells and to the dendritic spines in hippocampal neurons, where it could be accessed by extracellularly applied specific fluorescent probes. The efficient targeting of the YFP-5-HT3A to the cytoplasmic membrane is in line with the large serotonin-elicited currents (nA range) measured by whole-cell voltage-clamp recordings in transfected HEK-293 cells. In contrast, alpha3beta4-nAChRs expressed in the same cells yielded weaker ACh-evoked responses. Taken together, the fluorescent and electrophysiological studies presented here demonstrate the predominant intracellular location of alpha3beta4-nACh receptors and the predominant expression of the 5-HT3AR in dendritic surface loci.