We undertook the present study to examine alterations affecting the RB pathway in the G1 checkpoint and to determine their potential clinical significance in children affected with nonfamilial retinoblastoma. Using immunohistochemistry, patterns of expression of pRB, p16/INK4A, and E2F1 were analyzed in tissue from a cohort of 86 well-characterized patients with nonfamilial retinoblastoma diagnosed at the "Instituto Nacional de Pediatria" in Mexico City. The relationship of these phenotypes to proliferative index was assessed by analysis of Ki67 antigen expression. pRB expression was found in 11 (13%) cases. Using a hypophosphorylated specific pRB antibody, we observed low levels of underphosphorylated pRB expression in only 1 of 9 evaluable positive cases. These data suggest that the detected pRB products were hyperphosphorylated and thus had decreased functional activity. Increased p16 nuclear expression was found in only 6 tumors. No tumors showed deletions or mobility shifts of the INK4A gene. Undetectable pRB levels were significantly associated with undetectable p16 expression (odds ratio, 10.8; 95% confidence interval, 1.4-81.3; P =.03). All tumors showed nuclear immunoreactivities for E2F1 and Ki67. Increased Ki67 proliferative index was associated with increased staining for E2F1 (r =.44; P =.008) and increasing clinical stage (P =.03). Among children with unilateral disease, the mean Ki67 proliferative index was significantly higher in children with advanced clinical disease (stages 3 and 4) (mean 81.25; SD 6.78) than in those with earlier stage disease (mean 69.50; SD 9.45) (P = 0.001). Among children with bilateral disease, however, the mean proliferative index was not significantly higher for children with advanced clinical stage. When examining all cases together, there was a significant trend toward increasing proliferative index with increasing clinical stage (P =.03). In unilateral tumors, we also found that presence of detectable pRB was associated with a lower percentage of cells expressing E2F1 (46.7% v 70.8%) (P = 0.05), whereas there was no association between presence of pRB and E2F1 among bilateral tumors. We have found that expression of some of the cell cycle markers examined varies according to laterality, suggesting underlying differences in the capacity for cell cycle regulation between these 2 forms of the disease. Differences in capacities for cell cycle regulation may account for some differences in clinical behavior. Thus, the inclusion of molecular markers may become useful adjuncts to clinicopathological staging and subsequent determination of therapy.