This study used human umbilical vein endothelial cells (HUVECs) that were treated with 17beta-estradiol for 5 days as 1h pulse or 24h continuous treatment at concentrations such that the 24h exposure (concentration x time) was identical in both conditions. Cell proliferation was studied and gene expression profiling was carried out using the Affymetrix GeneChip microarray analysis. Changes in morphology and apoptosis in HUVECs were examined with electron microscopy. Time-course studies of expression of genes vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were performed by quantitative PCR. We observed that cell proliferation was significantly decreased over days 3-5 with pulsed estradiol treatment relative to constant exposure. Microarray results showed that after 5 days, 801 genes differed (P<0.05) between continuous versus pulsed estradiol treatment. Functional analysis showed a significant number of genes to be associated with apoptosis and cell cycle pathways. We did not find any evidence of apoptosis from flow cytometry or electron microscopy examination. Our study highlights a large number of significantly different molecular responses to estradiol depending upon the mode of administration of estradiol. Significant changes were observed in genes involved in apoptosis and proliferation including VEGF, IGF receptors, and tumor protein p53.