To better understand the role of the transient expression of ryanodine receptor type-3 (RyR3) on Ca²⁺ homeostasis during development of skeletal muscle we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at MOI 0.2, 0.4, 1.0 and 4.0 and analysis of their pattern of expression, caffeine sensitivity and resting free Ca²⁺ concentration ([Ca²⁺]r) were performed. Although increased MOIs resulted in an increased expression of each receptor isoform, it did not significantly affect the immuno-pattern of RyRs, or the expression levels of calsequestrin, triadin or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca²⁺]r and lower caffeine EC₅₀s than cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher resting Ca²⁺ in RyR3 expressing cells, studies of [³H]-ryanodine binding demonstrated that there are intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of post-natal development exhibited a transient increase in [Ca²⁺]r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca²⁺]r than adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca²⁺]r compared to RyR1 at any expression level. These data suggest that in the co-expression of RyR1 and RyR3 at different levels may constitute a novel mechanism to regulate [Ca²⁺]r in skeletal muscle.