We reported previously that chick myocardial cells placed into monolayer cell culture lost tetrodotoxin (TTX) sensitivity when tested at 72 h. To further characterize the change, ventricular myocardial cells were dispersed from chick embryos 14–16 days old; these hearts are TTX-sensitive before dispersal. Intracellular microelectrode penetrations were made into spontaneously beating cells at 9–72 h after culturing. No TTX-sensitive cells were found. Spontaneous action potentials with concomitant contractions continued in the presence of TTX (8 μg/ml), and the maximum rate of rise of the action potentials (+V̇ max) (control of 2–20 V/sec) was not reduced. Since the cells did not adhere to the vessel before 9 h, suspensions of cells were studied 1–8 h after dispersal to determine the rapidity of the loss of TTX sensitivity; all cells which contracted spontaneously or responded to electrical stimulation continued to beat in TTX. Addition of cycloheximide or actinomycin D did not prevent the loss of TTX sensitivity. The loss is not due to the use of trypsin (0.01 %) because dispersal by collagenase also resulted in loss of TTX sensitivity. Furthermore, cells separated mechanically (from 8-day-old hearts) also lost TTX sensitivity. In addition, loss of TTX sensitivity did not occur in frog sartorius muscles organ cultured for several days in 0.01 % trypsin. The loss of TTX sensitivity occurred even in multilayered cell cultures. Chronic exposure to carbachol or isoproterenol did not prevent the loss. However, elevation of K + in the medium (12–60 mM) prevented or reversed the loss of TTX sensitivity in some cells (~50 %), although +V̇ max remained low. Hence, the loss of TTX-sensitive fast Na + channels upon cell dispersal ( a) occurs very rapidly (less than 60 min), ( b) is not due to the use of trypsin, ( c) is independent of protein synthesis, ( d) is not solely a function of cell association, ( e) is not influenced by neurotransmitters, and ( f) is prevented or reversed by culturing in elevated [K +] 0. The mechanism of the changes in characteristics of the cation channels remains to be elucidated.