Rana temporaria sartorius muscle fibres were exposed to varied sequences of solution and temperature changes that have been employed hitherto in procedures that sought to decouple the transverse tubules from the surface membrane. The incidence of such detubulation was assessed in large numbers of fibres through demonstrating a loss or otherwise of the after-depolarization that normally reflects successful tubular propagation of the surface action potential. This criterion yielded assessments of the existing detubulation techniques in agreement with earlier results. The experiments then developed an improved detubulation procedure that required only brief (15 min) exposures to glycerol, its replacement in a single step by a Ca2+/Mg(2+)-Ringer solution for 30 min, and rapid cooling from room temperature (19-21 degrees C) to 6-10 degrees C prior to final restoration of the normal Ringer solution. This sequence of steps yielded an optimal incidence (98%) of detubulation in viable surface fibres that were amenable to electrophysiological studies. Studies that systematically modified the detubulation procedure demonstrated that the omission of any one step in the protocol significantly reduced the incidence of detubulation with or without accompanying deteriorations in fibre resting potentials. Successful detubulation accordingly required an initial exposure to an optimal glycerol concentration that lasted for a minimal duration and for its abrupt withdrawal. Inclusion of a cooling step within 30 min after glycerol withdrawal was coincident with, and critical to, optimal tubular isolation. Thus, cooling steps that either preceded, or that followed the glycerol withdrawal step by more than 60 min, resulted in a sharp reduction in the incidence of detubulation. Similarly, a critical period of exposure to Ca2+/Mg2+ Ringer solution also promoted detubulation without compromising the recovery of stable and satisfactory resting potentials. The findings reported here remain consistent with a primarily osmotic mechanism for detubulation. However, they demonstrated additional and important influences of temperature and of divalent cation concentration on the extent of tubular detachment when such factors were modified during the time course of the expected volume changes that followed each adjustment in osmotic condition.