The free intracellular calcium concentration, [Ca2+]i, was studied in single myotubes using the fluorescent Ca2+ indicator fura-2. Myotubes cultured from satellite cells of small muscle specimens from Duchenne muscular dystrophy (DMD) patients were compared with human control myotubes and with myotubes cultured from MDX and control mouse muscle satellite cells. The resting [Ca2+]i levels in DMD and control myotubes were not significantly different, i.e. 104 +/- 26 nM (mean +/- SD, n = 190 cells from eight DMD patients) compared with 97 +/- 25 nM (175/seven controls) and were not significantly lower than the corresponding murine values (154 +/- 33 nM, n = 135 MDX myotubes; 159 +/- 34 nM, n = 135 controls). All myotubes reacted to 10 microM acetylcholine or 40 mM KCl with fast transient increases of [Ca2+]i. After application of a hyposmotic (130 mOsm) solution, [Ca2+]i was increased 1.5- to 3-fold within 2-3 min, the DMD myotubes tending to stronger reactions (significantly higher [Ca2+]i in 2 out of 6 cases). The response was usually transient, [Ca2+]i decreasing to the initial level within 10 min. Gadolinium (50 microM) reduced the response by 50%-70%, indicating that the osmotic shock increased Ca2+ influx. During exposure to high (15 mM) [Ca2+]e, [Ca2+]i of DMD and control cells was 1.5- to 2-fold higher. Adult muscle fibres from MDX mice and controls showed identical Ca2+ resting levels (n = 45 fibres from three mice in each case), but did not respond to decreased external osmolarity with a change in [Ca2+]i. The results indicate that lack of dystrophin in muscle fibres does not necessarily lead to increased [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)