A new marker for cells of the osteoblastic lineage was identified by raising monoclonal antibodies against an immortalized rat osteoblastic cell line. Among the different antibodies one was selected which, on tissue sections, strongly reacts with osteoblasts, preosteocytes, and osteocytes. This antibody, designated E11, recognizes an antigen localized at the cell surface. The cDNA encoding the E11 antigen was cloned from a cDNA library prepared from ROS 17/2.8 cells, using a eukaryotic expression system. The Ell cDNA sequence revealed homology with the murine OTS-8/gp38 sequence. In situ hybridization confirmed that E11 mRNA expression in bone is restricted to osteoblasts and osteocytes. The tissue specificity of the E11 expression was studied by immunohistochemistry and Northern blot analysis. Apart from bone, E11-positive cells were also found in lung: namely, the alveolar cells of type I. Epithelial cells of the choroid plexus and endothelial cells of lymphatic vessels were also labeled with mAb E11. These results were confirmed by Northern blot, as the 1.8 kb Ell mRNA transcript was detected in bone and also in lung, brain, and skin. In conclusion, we describe a novel osteoblastic product which is expressed by mature osteoblasts and newly formed osteocytes.