In this study we used adenovirus vector-mediated transduction of either the p53 gene (rAd-p53) or the p21(WAF1/CIP1) gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21(WAF1/CIP1) within a human ovarian cancer cell line, 2774, and the derivative cell lines, 2774qw1 and 2774qw2. We observed that rAd-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells. Surprisingly, overexpression of p21(WAF1/CIP1) also triggered apoptosis within these two cell lines. Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX, BCL2, and caspase 3 genes, specific in rAd-p53-induced apoptotic cells, was not altered in rAd-p21-induced apoptotic cells, suggesting p21(WAF1/CIP1)-induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells infected with rAd-p21 on 60,000 cDNA microarrays identified 159 genes in response to p21(WAF1/CIP1) expression in at least one time point with 2.5-fold change as a cutoff. Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21(WAF1/CIP1) and 93 genes in response to p21(WAF1/CIP1) expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways, and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21(WAF1/CIP1)-induced apoptosis. Furthermore, promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21(WAF1/CIP1). This study has profound significance toward understanding the role of p21(WAF1/CIP1) in p53-independent apoptosis.