To explore the function of the giant AHNAK molecule, first described in 1992 [Shtivelman, E., Cohen, F. E. & Bishop, J. M. (1992) Proc. Natl. Acad. Sci. USA 89, 5472-5476], we created AHNAK null mice by homologous recombination. Homozygous knockouts showed no obvious phenotype, but revealed instead a second AHNAK-like molecule, provisionally designated AHNAK2. Like the original AHNAK, AHNAK2 is a 600-kDa protein composed of a large number of highly conserved repeat segments. Structural predictions suggest that the repeat segments of both AHNAKs may have as their basic framework a series of linked, antiparallel beta-strands similar to those found in beta-propeller proteins. Both AHNAKs appear to localize to Z-band regions of mouse cardiomyocytes and cosediment with membrane vesicles containing the dihydropyridine receptor, which is consistent with earlier reports that the AHNAKs are linked to L-type calcium channels and can be phosphorylated by protein kinase A. The localization of the AHNAKs in close proximity to transverse tubule membranes and Z-band regions of cardiac sarcomeres raise the possibility that they might be involved in regulating excitation/contraction coupling of cardiomyocytes, but other studies indicate that the association of AHNAKs with calcium channel proteins is more widespread. AHNAK2 is predicted to have a PDZ domain within its N-terminal, nonrepeating domain, which may mediate these interactions.