The hemolymph-like HL3 saline(Stewart et al., 1994)and standard saline(Jan & Jan, 1976)are two widely used bathing solutions for physiological recordings at the Drosophila larval neuromuscular junction. It has been established that longevity of larval preparations is better maintained in HL3 saline. However, HL3 can produce results that are inconsistent with previous findings in standard saline, particularly on temperature sensitivity and membrane excitability phenotypes. In wild-type larvae, the excitatory junctional potentials(EJPs)in standard saline(containing 4 mM Mg(2+)and 1.8 mM Ca(2+))were not blocked by a temperature increase up to 39-40 degrees C, consistent with unimpaired larval locomotion below these temperatures. However, in HL3 saline(containing 20 mM Mg(2+)and 1.5 mM Ca(2+)), EJPs were blocked at 30 degrees C. As for temperature-sensitive mutants nap(ts)and para(ts), the EJP-blocking temperatures were decreased from about 29 and 33 degrees C in standard saline to about 23 and 26 degrees C in HL3, respectively. Compound action potential recordings confirmed that segmental nerve action potentials were more readily blocked by a temperature increase in HL3 than in standard saline. Axonal excitability was suppressed in HL3 even at room temperatures, as evidenced by a lengthened refractory period in wild-type larvae. Similar suppression occurred for the hyper-excitable double mutant eag Sh, which maintained high-frequency spontaneous EJPs in standard saline but showed a rapidly declining EJP frequency in HL3. Application of HL3 saline also strongly suppressed the prolonged transmitter release following removal of repolarization mechanisms by K(+)channel blockers or by the eag Sh mutation previously described in standard saline. These discrepancies suggest that the high divalent cation content in HL3 may confer a surface charge screening effect to suppress nerve membrane excitability. We found that a minimal adjustment of the HL3 saline, decreasing the Mg(2+)ion concentration from 20 to 4 mM, was sufficient to resolve the discrepancies. While retaining the longevity of the larval neuromuscular preparation, the modified HL3 saline(HL3.1)restored the established wild-type EJP properties as well as phenotypes of several widely used temperature-sensitive and hyper-excitable mutants previously documented in standard saline.