Using the new signal sequence trap (SST) method, we isolated several clones encoding secreted and transmembrane proteins from KUSA cells, a murine osteoblast-like cell line. One isolated novel clone, termed entactin-2, exhibited a high similarity to mouse entactin/nidogen, a basement membrane protein. Although deduction of the amino acid sequence of entactin-2 revealed only 27.4% homology to entactin, many structural similarities were seen between both proteins. Entactin-2 contains five EGF-like and two thyroglobulin-like motifs, which are both cysteine-rich. Comparison of both proteins clearly revealed that entactin-2 also contains related domain structures. The rod-like domain of entactin-2, containing the RGD integrin recognition sequence, fused to glutathione-S transferase (GST), revealed a cell surface-binding activity similar to that of entactin. In addition, the tissue distribution of entactin-2 mRNA resembled that of entactin. Furthermore, mRNA expression of both genes decreased as osteoblastic differentiation progressed. These results suggest that entactin-2 is a member of the entactin gene family, may have entactin-related functions, and might act as a basement membrane component.