In this study, we have biochemically characterized the high-stearic sunflower (Helianthus annuus L.) mutant CAS-14. This mutant displays an abnormal fatty acid composition along the length of the seed when grown at high temperatures. Thus, at the expense of oleate and linoleate, CAS-14 seeds present an increasing axial gradient of stearic acid from the embryo to the distal extreme of the seed. The accumulation of oil in this mutant was initially characterized by analysing the incorporation of radiolabelled acetate, a fatty acid synthetic precursor, into the developing seed tissues in vivo. These experiments indicated that the mutant phenotype was associated with a decrease in the soluble stearoyl-acyl carrier protein desaturase (SAD) activity, as later confirmed when assessing this activity in cell-free extracts from developing sunflower kernels. Furthermore, SAD enzyme gene transcription was also examined in this tissue, identifying the coordinated decrease in the transcription of the sad17 and sad6 genes as underlying the decrease in enzyme activity. On the basis of these results and those previously obtained on the inheritance of the CAS-14 trait, we discuss the possible regulatory mechanisms acting on plant soluble desaturases.