Mesothelial cells are involved in a variety of biological processes, which include the formation of peritoneal adhesions. The cultures of human peritoneal mesothelial cells comprise an important tool to investigate the behavior and functions of mesothelial cells. Very little is known about the differences among mesothelial cells isolated from different sources and about the changes in specific functions as caused by cell propagation in vitro or that result from storage of cells at low temperatures. This study aims to characterize 2 particular cellular activities relevant for tissue repair, which include the expression of intercellular adhesion molecule-1 (ICAM-1) and the gelatinase activity; in addition, this study will assess the effect of hyaluronan, which is an antiadhesive agent, on these cellular activities. Viable cell lines were established from both omentum and peritoneal lavage fluid from 7 patients. Both ICAM-1 expression, which was measured by enzyme-linked immunosorbent assay (ELISA), and matrix metalloproteinase (MMP) bioactivity, which was measured by zymography, were measured in the 2nd and 4th passage; the latter also was measured after freezing and storing of cells in liquid nitrogen. The effects of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), phorbol myristate acetate (PMA), and hyaluronan were analyzed. ICAM-1 was constitutively expressed and stimulated by IL-1β, TNF-α, and PMA. All cell lines produced both MMP-2 and MMP-9. Only the latter activity was affected by TNF-α and, especially so, PMA. Differences were found between the 2nd and 4th passage, as well as between cells of different lineage, mostly so if the relative stimulation by the various agents was compared. The addition of sodium hyaluronate either to control cultures or to cultures together with any of the 3 stimuli examined did not significantly change either ICAM-1 expression or gelatinase activity. The freezing and storage of cells did not affect their functions. Both the human omentum and peritoneal lavage fluid are good sources to establish mesothelial cell lines, which can be propagated also after freezing without qualitative changes in their ability to express ICAM-1 and produce the gelatinases. For omental cells, a differential effect of stimulation occurs depending on whether the cells have been passaged 2 or 4 times. The presence of hyaluronan did not affect the expression of ICAM-1 or the gelatinases.