To characterize responses of the left ventricle (LV) to pressure overload at the genomic level, we performed high-density microarray analysis on individual mouse LVs. Male and female mice underwent transverse aortic constriction. At 1 day and 30 wk, the LV free wall was harvested and RNA isolated from 27 individual ventricles was analyzed on Mu74Av2 GeneChips, which contain approximately 12,483 distinct genes. Interestingly, a greater number of genes was regulated in response to acute overload than in response to chronic overload. Hierarchical cluster analysis revealed the presence of several distinct expression profiles. Of these clusters, the majority contained genes that were regulated either in response to acute overload or both acute and chronic overload. In addition, clusters revealing sex-specific responses to overload were detected. In summary, the acute and chronic genomic responses to pressure overload are distinct. Moreover, sex modifies these responses. Furthermore, these studies have uncovered several novel and potentially important genes that are regulated in response to overload and may open unrecognized avenues for further functional analysis.