A self-inactivating CD-tagging retroviral vector was used to introduce epitope and GFP tags into genes and proteins in NIH 3T3 cells. Several hundred cell clones, each expressing GFP fluorescence in a distinctive pattern, were isolated. Molecular analysis showed that a wide variety of genes and proteins, some known and some newly discovered, had been tagged. The analysis also revealed that, in the great majority of instances, the abundance and cellular location of the tagged protein mirrored that of its untagged counterpart. This approach provides a systematic means for the functional annotation of mammalian genomes and proteomes in living cells.