The authors compared detection of proliferating vascular smooth muscle cells (SMC) in vitro and in vivo with proliferating cell nuclear antigen (PCNA) immunohistochemistry and two established methods: [3H]thymidine autoradiography and bromodeoxyuridine immunohistochemistry. Labeling with [3H]thymidine and bromodeoxyuridine of rat vascular SMC in culture stained 11% +/- 2% and 11% +/- 1% of cells, and PCNA immunohistochemistry 22% +/- 2% of cells. Proliferation in the media of the denuded rat aortae was highest 3 days after denudation: 4.2%, 3.8%, and 4.7% of labeled cells with [3H]thymidine, bromodeoxyuridine, and PCNA, respectively. In the intima, proliferation was highest 7 days after denudation with 42% [3H]thymidine, 40% bromodeoxyuridine, and 46% PCNA-positive cells. With double labeling, all [3H]thymidine-positive cells were PCNA positive, whereas some cells were only positive for PCNA. The authors conclude that PCNA immunohistochemistry compares favorably with [3H]thymidine autoradiography, and bromodeoxyuridine immunohistochemistry.