W e investigated the in vivo introduction of a reporter gene into healing rat patellar ligaments using the hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer method. The mid-portion of the medial half of the patellar ligament was cut transversely with a scalpel in 14-wk-old male Wistar rats. A HVJ-liposome suspension containing beta-galactosidase (beta-gal) cDNA was injected directly into the injured site and pooled in the fascial pocket covering the injured site 3 d postoperatively. Thereafter, beta-gal-labeled cells were observed in the wound site accounting for 3% of the wound cells on the first day, 2% on the third, 7% on the seventh, 6% on the 14th, 2% on the 28th, and 0.2% on the 56th day after injection. The beta-gal-labeled cells were initially localized in and adjacent to the wound site, but they were observed spreading into the ligament substance away from the wound on the seventh day after injection. On day 28, beta-gal-labeled cells were observed throughout the length of the ligament substance. With double-labeling for marker antigens for monocyte/macrophage (ED-1) and for collagen I aminopropeptide (pN collagen I), it was revealed that fibroblastic (pN collagen I-positive) cells accounted for 63% and monocyte/macrophage lineage cells for 32% of the beta-gal-labeled cells in the day 7 wound. On day 28, they formed 58 and 35% of the beta-gal-labeled cells in the wound, respectively. Thus, we succeeded in introducing the beta-gal gene into healing rat patellar ligament. Moreover, labeling of the transfected cells made it possible to identify a biological event, namely that the cells in and around the wound site infiltrate into the uninjured ligament substance and come to populate the whole length of the ligament substance as repair progresses. These results suggest that ligament healing may involve not only the repair of the wound site itself but also extensive cellular infiltration of ligament substance adjacent to the wound.