To investigate the recombinational machinery of mammalian cells, we have constructed plasmids that can be used as substrates for homologous recombination. These plasmids contain two truncated nontandem, but overlapping, segments of the neomycin resistance gene, separated by the transcription unit for the xanthine guanine phosphoribosyl transferase gene. Recombination between the two nonfunctional neomycin gene sequences generates an intact neomycin resistance gene that is functional in both bacteria and mammalian cells. Using these plasmid substrates, we have characterized the frequencies and products of recombination events that occur in mouse 3T6 cells soon after transfection and also after stable integration of these DNAs. Among the chromosomal recombination events, we have characterized apparent deletion events that can be accounted for by intrachromatid recombination or unequal sister chromatid exchanges. Other recombination events like chromosomal inversions and possible gene conversion events in an amplification unit are also described.