We analyzed contractile function of ventricular myocytes derived from young (2-4 months) and aged mice (24-26 months). Isolated myocytes were continuously stimulated by voltage clamp pulses from -80 to -45 mV (prepulse, 10 ms) immedi- ately followed by a voltage step to 0 mV (160 ms) at 1Hz (37°C). Calcium transients were analyzed (Indo-1 fluorescence), contractions were evaluated (sarcomere shortening) and myofilament ATPase activity was determined (malachite green assay). Furthermore, expression of calcium handling proteins, NOS isoforms and NADPH oxidase subunits was quantified by real- time PCR or Western blots. We found that aged myocytes showed decelerated shortening/ relengthening without changes in fractional shortening. Calcium transient decay was similarly decelerated, but the amplitude of calcium transients was increased with aging. Calcium sensitivity of myofilaments of aged myocytes was reduced. These age-dependent alterations occurred without changes in calcium handling protein expression but were reversed by the superoxide scavenger tiron. NADPH oxidase and NOS1 that are putative sources of superoxide and nitric oxide were upregulated in aged hearts. Accordingly, contraction of aged myocytes was accelerated by inhibitors of NADPH oxidase (apocynin, DPI) or the NO scavenger PTIO. NOS1 deficient mice showed no alterations in calcium transients and contractile function with aging. We conclude that superoxide and nitric oxide derived from age-dependently upregulated NADPH oxidase and NOS1 contribute to the contractile dysfunction of aged murine ventricular myocytes. These reactive oxygen and nitrogen species modulate the calcium homeostasis of aged myocytes, but also interfere with the function of myofilaments, i.e. by modulation of calcium sensitivity.