Methyl C18 polyenoate concentrates were prepared from two samples of butter fat by low temperature crystallization and fractional distillation. The concentrates were fractionated on a silicic acid column and the resulting fractions were analyzed by ultraviolet and infrared spectrophotometric methods. About 42 and 30% repectively, of the non‐conjugated dienoate in the two samples were shown to have thecis,trans configuration. Fractions rich in dienoate and trienoate were prepared from the C18 polyene concentrates by silicic acid chromatography and the nature of these acids was studied by bromination, lipoxidase enzyme methods, and by alkali isomerization for varying periods of time. About 65 and 73%, respectively, of the non‐conjugated dienoate in the two samples investigated were found to consist of linoleic acid while 79 and 71% of the trienoate were linolenic acid. Linoleic and linolenic acids were identified by preparing the characteristic tetra‐ and hexabromostearic acids. Atrans,trans isomer of linoleic acid does not seem to be present in butter fat. A major proportion of the non‐conjugated dienoic acid other than linoleic acid was found to have widely separated double bonds withcis,trans configuration. Occurrences of a C15 saturated acid, a branchedchain C17 saturated acid, and a heptadecenoic acid were indicated by gas chromatography.