LINE-1 retrotransposons (L1s) constitute approximately 17% of human DNA, and their activity continues to affect genome evolution. Retrotransposition-competent human L1s encode two proteins required for their mobility (ORF1p and ORF2p); however, biochemical activities associated with ORF2p have been difficult to detect in cells. Here, we show for the first time the colocalization of L1 RNA, ORF1p and ORF2p to a putative ribonucleoprotein retrotransposition intermediate. We further demonstrate that ORF2p preferentially uses its encoding RNA as a template for reverse transcription. Thus, our data provide the first biochemical evidence supporting the cis-preferential action of the L1 reverse transcriptase.