Regulation of the density of voltage-dependent Na+ channels (VDNC) was studied in chick myotubes during their development in culture. The density of VDNC was assessed quantitatively in terms of the maximum rate of rise (M.R.R.) of Na+ spikes. Chronic treatment of myotubes, whose density of VDNC had reached a plateau level after 6 days in culture, with the calmodulin (CaM) inhibitor W-7 caused a further increase in the density. A derivative of W-7, known as W-5, which has a lower affinity for CaM than does W-7, was without effect. A selective inhibitor of Ca2+/CaM-dependent protein kinase II (CaM-KII), namely, KN-62, also caused an increase in the density of VDNC when its effect was examined in mature myotubes. A structural analogue of KN-62, namely, KN-04, which is a much less effective inhibitor of CaM-KII, was without effect. These results suggest that density of VDNC in developing myotubes is regulated by Ca2+/CaM and CaM-KII. However, the role of CaM-KII in this regulation is not straightforward since it appears to decrease the density of VDNC in mature myotubes, but to increase their density in immature myotubes.