Cell lines, such as those of teratocarcinoma and embryonic stem cells, fail to support high G418 resistance after transfection of neo vectors. To alleviate this, we modified pSV2-neo in two steps, first with tandem promoters of SV40 early genes and HSVtk, then by removing an improper met codon located immediately upstream of the authentic initiator codon in the mRNA sequence. In the final product, pSTneoB, the neo transcription unit has Xhol sites at both ends. pSTneoB yields G418-resistant transformants of teratocarcinoma cells at dramatically higher efficiencies than pSV2-neo. This vector extends the application of G418 selection in gene transfer to a wider range of cell types.