Human epithelial cell lines derived from carcinomatous and nonmalignant tissues were characterized with respect to the presence and distribution of fibronectin by immunofluorescence microscopy. In cell lines derived from nonmalignant tissues or from primary carcinomas, fibronectin was found predominantly in an extracellular matrix. Cell lines derived from metastatic carcinomas displayed very little or no fibronectin. Metabolic labeling studies indicated that a positive line synthesized fibronectin de novo rather than absorbing the protein from the media. Negative lines neither synthesized fibronectin nor secreted it into the culture fluid, suggesting that they were not producing fibronectin. Cells in culture seem to change their properties after extensive subculture since a small amount of fibronectin in an extracellular matrix was observed after extensive subculture of 2 metastatic lines that were originally negative.