Rat conjunctival goblet cells were stimulated with EGF for 16, 18, and 24 hours, the times at which cell proliferation had just begun, was intermediate, or was near completion, respectively. The percentage of cells that had entered the specific phases of the cell cycle was determined. Subsequently, the percentage of cells in each phase of the cell cycle that contained pERK translocated to the nucleus was measured. At 16 hours, 55% ± 12% of cells were in the G
1 phase with smaller percentages of cells in the S, G
2, and M phases
(Fig. 6A) . At this time 46% ± 23% of cells in G
1 contained translocated pERK. As the cells progressed farther into the cell cycle, a smaller percentage of proliferating cells contained pERK, until at the M phase no proliferating cells contained pERK. At 18 hours of stimulation, more cells were in each stage of the cell cycle compared to 16 hours
(Fig. 6B) . A larger percentage of cells in the G
1 phase, 60% ± 21%, contained translocated pERK than at 16 hours. The percentage of cells in the M phase containing pERK in the nucleus increased to 9% ± 9% compared with 0% of cells at 16 hours. At 24 hours of stimulation, the percentage of cells in G
1 decreased to 27% ± 7%, whereas 18% ± 4%, 32% ± 2%, and 25% ± 4% of the cells were in S, G
2, and M, respectively. These levels indicate that the cells were progressing through the cell cycle and dividing
(Fig. 6C) . At this time, fewer cells in the G
1 and S phases contained translocated pERK (32% ± 9% and 31% ± 6%), but more cells in the G
2 and M phases (20% ± 6% and 17% ± 10%, respectively) contained translocated pERK compared with 16 and 18 hours. Thus, as cells enter the cell cycle, a larger portion of them contain pERK translocated to the nucleus and activated. As stimulation with EGF is prolonged, a larger percentage of cells with activated ERK in the nucleus is observed. These findings are consistent with a major role for nuclear, activated ERK in EGF stimulation of goblet cell proliferation.