This study investigates the role of claudin-1 and claudin-2 in forming tight junction (TJ) strands in fibroblasts. The researchers introduced cDNAs of claudin-1, claudin-2, and occludin into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy showed that both claudin-1 and claudin-2 were concentrated at cell contact sites through homophilic interactions, forming well-developed TJ-like strand networks. Freeze-fracture replicas confirmed that these strands were specifically labeled with anti-FLAG mAb. In contrast, occludin formed only a small number of short strands, though it was also concentrated at cell contact sites. However, when co-transfected with claudin-1, occludin was incorporated into claudin-1-based strands. These findings suggest that claudin-1 and claudin-2 are primarily responsible for TJ strand formation, while occludin functions as an accessory protein. The study provides a new method for analyzing TJ structure and function by reconstituting TJ strands in fibroblasts. The results indicate that claudin-1 and claudin-2, with four transmembrane domains, are key structural components of TJ strands, while occludin, though also present, plays a secondary role. The study also highlights the importance of homophilic interactions in the formation of TJ strands and the potential roles of claudins and occludin in regulating TJ function.This study investigates the role of claudin-1 and claudin-2 in forming tight junction (TJ) strands in fibroblasts. The researchers introduced cDNAs of claudin-1, claudin-2, and occludin into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy showed that both claudin-1 and claudin-2 were concentrated at cell contact sites through homophilic interactions, forming well-developed TJ-like strand networks. Freeze-fracture replicas confirmed that these strands were specifically labeled with anti-FLAG mAb. In contrast, occludin formed only a small number of short strands, though it was also concentrated at cell contact sites. However, when co-transfected with claudin-1, occludin was incorporated into claudin-1-based strands. These findings suggest that claudin-1 and claudin-2 are primarily responsible for TJ strand formation, while occludin functions as an accessory protein. The study provides a new method for analyzing TJ structure and function by reconstituting TJ strands in fibroblasts. The results indicate that claudin-1 and claudin-2, with four transmembrane domains, are key structural components of TJ strands, while occludin, though also present, plays a secondary role. The study also highlights the importance of homophilic interactions in the formation of TJ strands and the potential roles of claudins and occludin in regulating TJ function.