The fundamental question in this study is concerned with whether the increase of unsaturated fatty acids in the cell membrane is a general response of certain thermotolerant strains or species when exposed to superoptimal temperatures, and in combination with other stresses, especially oxidative stress. A strain of Lactobacillus helveticus, a species widely used as a starter in the dairy industry and able to tolerate high temperature and NaCl concentrations as well as acidic conditions, was chosen for this study. Cells of strain CNBL 1156, grown in its natural medium (i.e. milk whey), were exposed for 100 min to sublethal combinations of temperature, NaCl, H2O2 and pH, modulated according to a Central Composite Design. The fatty acid composition of cell lipid extract was identified by GC/MS. Polynomial equations, able to describe the individual interactive and quadratic effects of the independent variables on cell fatty acid composition, were obtained. The results and the mathematical models relative to the individual fatty acids indirectly suggest that desaturase activation or hyperinduction play an important role in the response to heat stress. In fact, the relative proportions of oleic, linoleic and palmitic acids increased with temperature in a range between 38 and 54 °C. The fatty acid profiles included vernolic acid (up to 37% of total fatty acids), an epoxide of linoleic acid not previously reported in microbial cells. In particular, this epoxide was present in cells exposed to low pH in combination with high temperatures and oxidative stress. In conclusion, these results provide experimental support to the hypothesis that the increase of an oxygen-consuming desaturase system, with a consequent increase in fatty acid desaturation, is a cellular response to environmental stresses able to protect the cells of this anaerobic micro-organism from toxic oxygen species and high temperatures.