The effect of calcium (0.5-6 mM) and neomycin (0.1-0.2 mM) on the maximum post-tetanic twitch tension (MTT) and post-tetanic depression (PTD) of the indirectly elicited twitch tension was studied on the mouse isolated phrenic nerve-diaphragm preparation. The effect of neomycin on MTT of directly stimulated twitch tension was also tested in (+)-tubocurarine pretreated preparations. Three-dimensional plots between MTT and frequency and duration of indirect tetanic stimulation revealed that the frequencies and durations inducing maximal MTT were 500 Hz for 20 s in 0.5 mM CaCl2, 100 Hz for 5 s in 2 mM CaCl2 and 100 Hz for 10 s in 6 mM CaCl2. The frequency and duration inducing maximal PTD was 100 Hz for 20 s in 0.5 mM CaCl2, but there was no PTD in 2 mM or 6 mM CaCl2. Neomycin was associated with significantly greater MTT than in control if the duration of tetanic stimulation was 1 or 2 s, while it was associated with less MTT if the duration of tetanic stimulation was 10 or 20 s. Neomycin caused PTD in 2 mM CaCl2; sometimes the depressive effect was so severe that twitch tension was abolished. The maximal depression effect was found after 100 Hz tetanic stimulation for 20 s. Increasing the extracellular calcium concentration to 6 mM antagonized the effects of neomycin on MTT and PTD, whereas neostigmine (1.6 microM) antagonized the effect partially. Neomycin had no effect on the MTT or PTD of the directly stimulated twitch tension. It is concluded that neomycin alters the conditions of tetanic stimulation inducing MTT.