Lentiviral vectors are valuable tools to express genes of interest in living animals and stem cell cultures. The use of promoters in lentiviral constructs has been successfully used to drive gene expression in particular cell types including neurons and glia of the central nervous system in vivo. However, their suitability in cell culture is less well documented. In this paper, we describe lentiviral vectors containing neuronal promoters of the murine stem cell virus, of the synapsin 1 gene, the tubulin alpha 1 gene, and the calmodulin kinase II gene, and the glial promoter of the glial fibrillary acidic protein gene to drive reporter gene expression in primary dissociated cerebellar cell cultures and in slice cultures. While the glial promoter was highly specific for glia, the neuronal promoters were active in neurons and glia of dissociated cultures to a comparable extent. In slice cultures, neuronal and glial promoters demonstrated higher, but not absolute selectivity for particular cell types. In addition, the promoters allowed for an efficient and graded expression of genes in dissociated cultures. By using selected combinations of vectors, it was also possible to drive the expression of two genes in one cell type with high efficiency. A gene of interest in combination with a reporter gene can thus be expressed in a graded manner to reveal gene function in a rather short time and in a complex cellular environment.