Previous studies have shown stimulatory effects of linoleic acid (LA, C18:2) on differentiation of rat muscle cells in culture (Allen et al.1985), but there appears to be little investigation of the effects of other fatty acids. The present study therefore compared the effects of different fatty acids on muscle cell differentiation in vitro. L6 myoblasts were cultured (Dulbecco's Modified Eagles Medium+10% fetal calf serum) in six-well plates until 80% confluent (day 0). Cells were then either harvested or the medium switched to differentiation medium (Dulbecco's Modified Eagles Medium+2% horse serum), supplemented with fatty acid or drug treatments. Cells were harvested on days 0–5 and assayed for creatine kinase (CK), protein and DNA contents, to give a measure of differentiation (CK/DNA). Initial studies indicated a stimulatory effect of the cis9trans11 (c9, t11) isomer of conjugated linoleic acid (CLA) relative to control. By contrast, the trans10, cis12 (t10, c12) isomer of CLA inhibited differentiation. Further experiments indicated that inhibition of differentiation by the t10, c12 CLA isomer was dose-dependent (up to 200μm) and may be via increased cell proliferation. LA and c9, t11 CLA stimulated differentiation at low concentrations (up to 50μm), but inhibited differentiation at high concentrations (200μm). In contrast, oleic acid stimulated differentiation at all concentrations, whereas the saturated fatty acid, palmitic acid, had no effect. The mechanism appeared not to involve either peroxisome proliferator-activated receptors α or γ. The data suggest that only unsaturated fatty acids have an effect and the presence or absence of a cis-9 double bond may be important.