An increase in the proportion of unsaturated fatty acids in milk is considered desirable for human health. A prerequisite for the manipulation of milk fat composition is a co-ordinated understanding of the complex interactions in its biosynthesis. It has been suggested that an increase in the expression of mammary stearoyl-CoA-desaturase (SCD) would enrich mono-unsaturated fatty acids in milk, and therefore improve its nutritional properties. To investigate the potential effects of changes in expression of mammary enzymes and substrate availability on milk fat composition, we constructed, parameterized and evaluated a mechanistic mathematical model of fatty acid biosynthesis and milk-fat triglyceride assembly. The objective was to describe changes in the amount and composition of milk fat produced by bovine mammary cells due to changes in nutrition. Using the model we found that a 50% up-regulation in SCD activity increased the molar fraction of milk triglyceride 18[ratio ]1 from 0·30 to 0·33 and 16[ratio ]1 from 0·04 to 0·06. Up-regulation of SCD therefore did not appear to be the optimal method for increasing the content of unsaturated fatty acids in milk fat. The model was also used to determine the likely rate-limiting processes for the incorporation of unsaturated fatty acids into milk fat. Halving the concentration of glycerol 3-phosphate increased the molar fraction of milk triglyceride 18[ratio ]1 from 0·30 to 0·35 and decreased the molar fraction of milk triglyceride 16[ratio ]0 from 0·30 to 0·22. This achieved the desirable outcome of producing more unsaturated low-fat milk. Our model also predicted that a K232A mutation in the bovine mammary DGAT1 gene that is linked with an increase in milk fat yield would be consistent with a 120% increase in the DGAT acylation rate and also would be associated with a decrease in milk mono-unsaturated fatty acids.