The endogenous production of unsaturated fatty acids (FA), particularly some monounsaturated FA (%MONO) and nearly all conjugated linoleic acids, is regulated by the Delta(9)-desaturase activity. The aims of this study were to assess the variation of this enzymatic activity within lactation, across dairy breeds, and to estimate its genetic parameters. The ratios of C14:1 cis-9 to C14:0, C16:1 cis-9 to C16:0, and C18:1 cis to C18:0 were calculated from FA contents predicted by mid-infrared spectrometry. Variance components and standard errors were estimated using average information REML. The multitrait mixed model included as fixed effects herd x test date x class of lactation number, class of days in milk x class of lactation number, class of age x class of lactation number, and regressions on breed composition. Four random effects were also included: animal genetic effect, 2 permanent environments (within and across lactations), and residual effect. Under the assumption that the calculated ratios are an approximate measurement of Delta(9)-desaturase activity, this study showed different sources of variation for this enzymatic activity. A slight difference was observed within lactation. The ratios of C14:1 cis-9 to C14:0 and C16:1 cis-9 to C16:0 increased as a function of days in milk. Differences across 7 dairy breeds were observed. The values of Delta(9)-desaturase indices observed for Jersey and Brown-Swiss cows were lower compared with Holstein. The opposite was observed for dual-purpose Belgian Blue cows. Values of heritability for the ratios of C14:1 cis-9 to C14:0, C16:1 cis-9 to C16:0, and C18:1 cis to C18:0 were 20, 20, and 3%, respectively. Negative genetic correlations observed between fat or protein contents and the 3 indices suggested that an increased activity of Delta(9)-desaturase could inhibit the synthesis of fat and protein in bovine milk. Negative correlations were also observed between fat or protein contents and the contents of 3 studied unsaturated FA in milk fat (C14:1 cis-9, C16:1 cis-9, and C18:1 cis). The positive genetic correlations observed between %MONO and the ratios of C14:1 cis-9 to C14:0 (0.72), C16:1 cis-9 to C16:0 (0.62), and C18:1 cis to C18:0 (0.97) showed that %MONO is linked to the Delta(9)-desaturase activity.