Adenovirus-mediated gene transfer into adult cardiac myocytes in primary culture is a potentially useful method to study the structure and function of the contractile apparatus. However, the consequences of adenovirus infection on the highly differentiated state of the cultured myocyte have not been determined. We report here a detailed analysis of myofilament structure and function over time in primary culture and after adenovirus infection. Adult rat ventricular myocytes in primary culture were infected with a recombinant adenovirus vector expressing either the LacZ or alkaline phosphatase reporter gene. Control and infected myocytes were collected at days 0-7 post-isolation/infection, and myofilament isoform expression was determined by SDS-PAGE and Western blot. Laser scanning densitometry showed that the α- to β-myosin heavy chain ratio, the stoichiometry of the myosin light chains and the expression of the adult troponin T isoform did not change over time in culture or with adenovinus treatment. Importantly, examination of Ca²⁺-activated tension in single myocytes showed no change in the shape or position of the tension-pCa relationship in the control and adenovirus infected myocytes during primary culture. These results indicate that the structure and function of adult cardiac myocytes are stable in short term primary culture and are not affected by adenovirus infection per se, and therefore provide the foundation for the use of adenovirus-mediated myofilament gene transfer to study contractile apparatus structure and function in adult cardiac myocytes.ain.