Virus-mediated gene transfer into neurons is a powerful tool for the analysis of neuronal structure and function. Recombinant sindbis virus has been previously used to study protein function in hippocampal neuron cultures as well as in hippocampal organotypic slice cultures. Nevertheless, some concern still exists about the physiological relevance of these cultured preparations. Acute hippocampal slices are a widely used preparation for the study of synaptic transmission, but currently recombinant gene delivery is usually achieved only through time-consuming transgenic techniques. In this study, we show that a subregion of the CA1 area in acute hippocampal slices can be specifically altered to express a gene of interest. A sindbis virus vector carrying an enhanced green fluorescent protein (EGFP) reporter was injected in vivo into the hippocampus of adult rats. After 18 h, rats were killed, and acute hippocampal slices, infected in the CA1 field, were analyzed morphologically and electrophysiologically. Infected slices showed healthy and stable electrophysiological responses as well as long-term potentiation. In addition, infected pyramidal cells were readily recognized in living slices by two-photon imaging. Specifically, the introduction of an EGFP-Actin fusion protein greatly enhanced the detection of fine processes and dendritic spines. We propose this technique as an efficient tool for studying gene function in adult hippocampal neurons.