Homozygous zebrafish of the mutant relaxed ( red ^ts25 ) are paralyzed and die within days after hatching. A significant reduction of intramembrane charge movements and the lack of depolarization-induced but not caffeine-induced Ca ²⁺ transients suggested a defect in the skeletal muscle dihydropyridine receptor (DHPR). Sequencing of DHPR cDNAs indicated that the α _1S subunit is normal, whereas the β _1a subunit harbors a single point mutation resulting in a premature stop. Quantitative RT-PCR revealed that the mutated gene is transcribed, but Western blot analysis and immunocytochemistry demonstrated the complete loss of the β _1a protein in mutant muscle. Thus, the immotile zebrafish relaxed is a β _1a -null mutant. Interestingly, immunocytochemistry showed correct triad targeting of the α _1S subunit in the absence of β _1a . Freeze-fracture analysis of the DHPR clusters in relaxed myotubes revealed an ≈2-fold reduction in cluster size with a normal density of DHPR particles within the clusters. Most importantly, DHPR particles in the junctional membranes of the immotile zebrafish mutant relaxed entirely lacked the normal arrangement in arrays of tetrads. Thus, our data indicate that the lack of the β _1a subunit does not prevent triad targeting of the DHPR α _1S subunit but precludes the skeletal muscle-specific arrangement of DHPR particles opposite the ryanodine receptor (RyR1). This defect properly explains the complete deficiency of skeletal muscle excitation-contraction coupling in β ₁ -null model organisms.