Three-dimensional skeletal muscle constructs engineered from myogenic cells isolated from the fast tibialis anterior (TA) muscle of rats have a 30% faster time to peak twitch tension (TPT) and 1/2 relaxation time (1/2RT) then constructs engineered from the slow soleus (SOL) muscle. As expected, the faster contraction and relaxation times for the TA constructs results in right shift of the force-frequency curve compared to those from the SOL. Fourteen days of chronic low frequency electrical stimulation (CLFS) slows the TPT 15% and the 1/2RT 14%, but does not alter the absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation results in an 80% increase in absolute force production with no change in TPT or 1/2RT. The addition of cyclosporine A (CsA) does not prevent the increase in force in SOL constructs following CLFS suggesting that calcineurin is not responsible for the increase in force. Furthermore, CsA increases the levels of fast MHC, force production and TPT but does not affect 1/2RT. Lastly, CLFS is able to reverse the CsA-induced shift towards fast MHC. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contract and relax slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation seen in engineered tissues.