The KPL2 gene is expressed predominantly in cells with cilia or flagella. We have previously demonstrated that a large intronic insertion in KPL2 is associated with immotile sperm cells and infertility in the domesticated pig (Sus scrofa). To fully characterize the structure of the mutation, we have now cloned and sequenced the insertion. The data identified the presence of a long interspersed nuclear element-1 (LINE-1) encoding all activities required for retrotransposition, including a 5'-untranslated region (UTR) with an internal RNA polymerase II promoter, two open reading frames (ORF1 and ORF2) separated by an intergenic region and a 3' UTR containing a polyadenylation signal. Characterization of the junctions between the LINE-1 and the genomic target revealed the presence of direct repeats of 14 bp at both ends, showing that integration occurred by target-primed reverse transcription. Furthermore, sequence analysis suggested that the aberrant splicing pattern of KPL2 transcripts induced by the LINE-1 element is caused by interference with putative intronic splice signals and activation of a cryptic splice site. These data demonstrate that integration of a transposition-competent L1 element into KPL2 is responsible for the defective spermatozoa, which accentuates the role of mobile DNA elements as insertional mutagens in mammalian genomes.