Lentiviral vectors have become very useful tools for transgene delivery. Based on their ability to transduce both dividing and nondividing cells and to produce long-term transgene expression, lentiviruses have found numerous applications in the biomedical sciences, including developmental neuroscience. This protocol describes how to prepare lentiviral vectors by calcium phosphate transfection and to concentrate viral particles by ultracentrifugation. Functional vector titers can then be determined by methods such as fluorescence-activated cell sorting or immunostaining. Effective titers in the range of 108 –109 infectious units/ml can be routinely obtained using these protocols. Finally, we describe the infection of primary neuronal cultures with lentiviral vectors resulting in 85–90 % cell transduction using appropriate multiplicities of infection.