The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β1a subunit. Lack of β1a results in (i) reduced membrane expression of the pore forming DHPR α1S subunit, (ii) elimination of α1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β1a from rather general functions of β isoforms. Zebrafish and mammalian β1a subunits quantitatively restored α1S triad targeting and charge movement as well as intracellular Ca2+ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β2a as the phylogenetically closest, and the ancestral housefly βM as the most distant isoform to β1a also completely recovered α1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca2+ transients and very limited tetrad formation compared with β1a. Consequently, larval motility was either only partially restored (β2a-injected larvae) or not restored at all (βM). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β1a isoform. Consequently, we postulate a model that presents β1a as an allosteric modifier of α1S conformation enabling skeletal muscle-type EC coupling.