Recently it was shown that several new pseudotyped adeno-associated virus (AAV) vectors support cardioselective expression of transgenes. The molecular mechanisms underlying this propensity for cardiac cell transduction are not well understood. We comparatively analyzed AAV vector attachment, internalization, intracellular trafficking, and nuclear uncoating of recombinant self-complementary (sc) AAV2.2 versus pseudotyped scAAV2.6 vectors expressing green fluorescence protein (GFP) in cells of cardiac origin. In cardiac-derived HL-1 cells and primary neonatal rat cardiomyocytes (PNCMs), expression of GFP increased rapidly after incubation with scAAV2.6-GFP, but remained low after scAAV2.2-GFP. Internalization of scAAV2.6-GFP was more efficient than that of scAAV2.2-GFP. Nuclear translocation was similarly efficient for both, but differential nuclear uncoating rates emerged as a key additional determinant of transduction: 30% of all scAAV2.6-GFP genomes translocated to the nucleus became uncoated within 48 h, but only 16% of scAAV2.2-GFP genomes. In contrast to this situation in cells of cardiac origin, scAAV2.2-GFP displayed more efficient internalization and similar (tumor cell line HeLa) or higher (human microvascular endothelial cell (HMEC)) uncoating rates than scAAV.2.6-GFP in non-cardiac cell types. In summary, both internalization and nuclear uncoating are key determinants of cardiac transduction by scAAV2.6 vectors. Any in vitro screening for the AAV pseudotype most suitable for cardiac gene therapy - which is desirable since it may allow significant reductions in vector load in upcoming clinical trials--needs to quantitate both key steps in transduction.