p19(ARF) is a tumor suppressor that leads to cell cycle arrest or apoptosis by stabilizing p53. p19(ARF) is not critical for cell cycle regulation under normal conditions, but loss of p19(ARF) is seen in many human cancers, and a murine p19(Arf) knockout model leads to malignant proliferation and tumor formation; its role in controlling nonmalignant proliferation is less defined. To examine this question, pulmonary artery smooth muscle cells (PASMC) were expanded in culture from a transgenic mouse in which the coding sequence of the p19(Arf) gene was replaced with a cDNA encoding green fluorescent protein (GFP), leaving the promoter intact. During the first 10 days in culture, wild-type, heterozygous, and knockout PASMC grew similarly, but, by day 14, p19(Arf)-deficient PASMC proliferated faster than p19(Arf) heterozygous or wild-type cells; reexpression of p19(Arf) prevented the increased proliferation. This time course correlated with activation of the p19(Arf) promoter, as indicated by the appearance of GFP positivity in p19(Arf)-deficient PASMC. By day 42, ∼80% of p19(Arf)-deficient cells were GFP-positive. When GFP-positive, p19(Arf)-deficient cells were sorted and subcultured separately, they remained GFP-positive, indicating that once cells had activated the p19(Arf) promoter, the promoter remained active in those and all subsequent daughter cells. In contrast, GFP-negative p19(Arf)-deficient cells gave rise to a combination of GFP-positive and -negative daughter cells over time. These results suggest that a subpopulation of PASMC are resistant to the signals that activate the p19(Arf) promoter, an event that would normally target these cells for arrest or cell death.