Ubiquitous ablation of nonmuscle myosin heavy chain II-B (NMHC II-B) in mice resulted in defects in the heart and brain, leading to lethality between embryonic day (E)14.5-E18.5. To understand the physiological role of NMHC II-B in the adult mouse heart, we used a Cre/loxP strategy to ablate NMHC II-B specifically in the heart by generating mice with exon 2 of Myh 10 flanked by loxP sites. These mice were then crossed with mice expressing Cre-recombinase under control of the alpha-cardiac MHC promoter, which is activated later in development than the NMHC II-B promoter. In contrast to conventional NMHC II-B ablated mice, 80% of the B-alpha-card/B-alpha-card mice survived to become adults. These mice developed a novel phenotype beginning at 6 months. EKG abnormalities including bradycardia, right axis deviation and a posterior hemiblock were noted. By 10 months, the echocardiogram of these mice showed a decreased fractional shortening of the left ventricle (21% vs 47% for wild-type mice) indicating a compromised cardiac function. Histopathological changes were seen by both light and EM microscopy in these hearts. At 6 months, an inflammatory cell infiltration was seen in the interstitum of all four chambers. By 10 months, changes included marked interstitial fibrosis in which large areas of the myocardium were replaced with connective tissue. Previously, we had shown that NMHC II-B was present in the Z-line and intercalated disks. Interestingly, both of these structures were found to be abnormal using EM. Most dramatic were the changes in the intercalated disks, which were markedly widened and distorted compared to wild-type controls. These results show that NMHC II-B, in addition to being required for normal cardiac development, also plays an important role in the adult mouse heart.