The lumbosacral spinal cords of 14.5-day gestation mice (E14.5) were ablated. The number of molecules of each of the four myogenic regulatory factor (MRF) mRNAs per nanogram of total RNA were evaluated in innervated and aneural fetal crural muscles. Accumulation of all four MRF mRNAs was affected in aneural muscle, but was never more than threefold different than in innervated muscles, considerably less than after adult denervation. The effect of the nerve varied with the MRF, the fetal age, and with the muscle (extensor digitorum longus muscle [EDL] vs. soleus muscle), with the nerve having multiple effects including down-regulation of certain MRF genes at specific periods (e.g., myoD and myogenin [E16.5–E18.5] and MRF4 in the EDL only [E18.5–E19.5]); limiting the up-regulation of certain genes, which occurred in the absence of innervation (e.g., myf-5 [E18.5–E19.5] and myogenin [E14.5–E16.5]); and even enhancing the accumulation of MRF4 mRNA (E14.5–E16.5). We hypothesize that factors other than nerve contribute to the down-regulation of myf-5 and myogenin mRNAs to adult levels. Innervation was required for the emergence of the slow, but not the fast, MRF mRNA profile at birth. MyoD, found in both the nuclear and cytoplasmic protein extracts of innervated fetal muscle, increased by ∼5-fold in the nuclear extracts (∼2.5-fold in the cytoplasmic) of E19.5 aneural muscles, significantly less than the 12-fold increase found in the nuclear extract of 4-day denervated adult muscle. This increase in aneural fetal muscle was due primarily to an increased concentration of myoD in muscle lineage nuclei, rather than to the presence of additional myoD+ muscle lineage nuclei. Developmental Dynamics 236:732–745, 2007. © 2007 Wiley-Liss, Inc.