Flectin, a protein previously described to be expressed in a left-dominant manner in the embryonic chick heart during looping, is a member of the nonmuscle myosin II (NMHC-II) protein class. During looping, both NMHC-IIA and NMHC-IIB are expressed in the mouse heart on embryonic day 9.5. The patterns of localization of NMHC-IIB, rather than NMHC-IIA in the mouse looping heart and in neural crest cells, are equivalent to what we reported previously for flectin. Expression of full-length human NMHC-IIA and -IIB in 10 T1/2 cells demonstrated that flectin antibody recognizes both isoforms. Electron microscopy revealed that flectin antibody localizes in short cardiomyocyte cell processes extending from the basal layer of the cardiomyocytes into the cardiac jelly. Flectin antibody also recognizes stress fibrils in the cardiac jelly in the mouse and chick heart; while NMHC-IIB antibody does not. Abnormally looping hearts of the NodalΔ 600 homozygous mouse embryos show decreased NMHC-IIB expression on both the mRNA and protein levels. These results document the characterization of flectin and extend the importance of NMHC-II and the cytoskeletal actomyosin complex to the mammalian heart and cardiac looping. Developmental Dynamics 237:3577–3590, 2008. © 2008 Wiley-Liss, Inc.