Whether long interspersed element-1 (L1 or LINE-1) retrotransposition can occur in quiescent, nondividing, and/or terminally differentiated somatic cells has remained an unanswered fundamental question in human genetics. Here, we used a ubiquitously active phosphoglycerate kinase-1 promoter to drive the expression of a highly active human L1 element from an adenovirus-L1 hybrid vector. This vector system achieved retrotransposition in up to 91% of actively growing immortalized cells, and we demonstrated that L1 retrotransposition can be suppressed by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine. This adenovirus vector enabled efficient delivery of the L1 element into differentiated primary human somatic cells and G1/S-arrested cells, resulting in retrotransposition in both cases; however, it was not detected in G0-arrested cells. Thus, these data indicate that L1 retrotransposition can occur in nondividing somatic cells.