Alpha-actinins belong to a family of actin-binding and crosslinking proteins and are expressed in many different cell types. Multiple isoforms of alpha-actinin are found in humans and are encoded by at least four distinct genes. Human skeletal muscle contains two sarcomeric isoforms, alpha-actinin-2 and -3. Previous studies have shown that the alpha-actinins function as anti-parallel homodimers but the question of heterodimer formation between two different isoforms expressed in the same cell type has not been explored. To address this issue, we expressed both alpha-actinin-2 and -3 in vitro and were able to detect their interaction by both blot overlay and co-immunoprecipitation methods. We were also able to demonstrate the presence of heterodimers in vivo in human skeletal muscle and in COS-1 cells transiently transfected with both isoforms. Our results clearly demonstrate the potential for alpha-actinin isoforms to form heterodimers which might have unique functional characteristics.