Although the liver is the major source of the adhesive glycoprotein vitronectin (Vn) in vivo, we recently demonstrated low levels of extrahepatic Vn transcription. In this report, in situ hybridization was employed to identify the Vn-producing cells at these extrahepatic sites. In the central nervous system (CNS), high levels of Vn transcripts were prominent in arachnoid cells and in cells frequently present in the vicinity of brain capillaries. Significant amounts of Vn mRNA were also detected in selected peripheral organs. In the myocardium, the signal was localized to cells in the endomysium and subepicardial fat. Additionally, the pulmonary alveolar walls contained Vn-positive cells. The parenchyma of the kidney and spleen were negative. Moreover, larger blood vessels and adjacent cells in the CNS and peripheral organs were devoid of the Vn transcript. Unexpectedly, the rate of Vn gene expression in subsets of cells present in the CNS was similar to that of hepatocytes. These results suggest that the low level of Vn gene expression detected by quantitative PCR may reflect relatively high levels of synthesis by a small subset of cells, and raise the possibility that tissue Vn may, in part, be derived from local biosynthesis rather than from plasma.