The isolation and culture of an enriched population of cells from rat lymph nodes that have several properties of high endothelial cells are described. High endothelial cells synthesize a unique sulphated glycolipid. This macromolecule in high endothelial cells was labelled with 35SO4 prior to cell isolation and was used to identify high endothelial cells after isolation. Collagenase digestion of pre-labelled lymph nodes yielded primary lymph node cultures in which two different cell types accounted for greater than 90% of non-lymphoid cells isolated. The majority (greater than 70%) were 20-30 microns diameter, round and 35S-labelled and were therefore high endothelial cells. The remaining unlabelled cells were 10-15 microns diameter and were identified as macrophages by phase-contrast microscopy. Isolated cells proliferated after 1-2 days and cultures were enriched for high endothelial cells as macrophages did not persist beyond 7-10 days. Small clumps (2-3 cells) of microvascular endothelial cells and/or adventitial fibroblasts were occasionally seen in primary cultures (angle 1% of isolated cells) but neither cell type proliferated. The identity of high endothelial cells was further substantiated using a polyclonal antiserum raised against lymph node cultures, which stained high endothelium in cryostat sections of lymph nodes. At confluence primary lymph node cultures bound lymphocytes as efficiently as high endothelium in lymphoid tissue and 40-fold more efficiently than rat aortic endothelial cells. It is concluded that lymph node cultures contain high endothelial cells and that these cells continue to express surface determinants for lymphocytes in vitro.