The isolated mammary glands of six lactating goats were perfused with heparinized and oxygenated blood for 8 to 11 h. Adequate quantities of glucose, acetate and amino acids (including valine) were added to the perfusate. Either unlabelled valerate or unlabelled isobutyrate was added in excess to the perfusate of one gland, while the respective symmetrical gland was used as a control. After the administration of valerate, the proportions of the odd-numbered fatty acids (C11:0, C13:0, C15:0) in the milk fat, collected every hour during perfusion, rose progressively after 5 h until the end. The synthesis of milk fatty acids from valerate is discussed. After isobutyrate was added to the perfusate, isoC12:O, isoC14:0 and isoC16:0 in the milk fat increased as compared to the control. The effect of isobutyrate indicated that valine acted as a precursor of milk iso-branched fatty acids after its metabolisation to isobutyryl-CoA. During perfusion in the presence of the complete substrate mixture, the proportion of certain major milk fatty acids (C10:0, C12:0, C14:0 and C16:0) increased, whereas the proportion of C18:0 and C18:1 decreased. These effects have been ascribed to the presence of acetate and beta-hydroxybutyrate in the substrate mixture.