Pasture-fed cattle show yellow pigmentation of their fat due to beta-carotene stored in this tissue. beta,beta'-Carotene-15,15'-oxygenase (betaCO) is an enzyme expressed in different tissues, and it cleaves beta-carotene into retinal. We compared the expression and activity of betaCO in duodenum and liver of cattle with pigmented or non-pigmented fat. In the duodenum, in situ hybridizations showed expression of betaCO in epithelial cells and crypts of the mucosa that was similar in animals from pigmented and non-pigmented fat; liver showed diffuse signal at lobules, but pigmented animals showed higher signals near the portal space. Analyses by real-time reverse-transcription polymerase chain reaction also showed amplification of mRNA for betaCO in duodenum and liver, with no difference between pigmented or non-pigmented animals. Enzyme activity was similar in the duodenum, but pigmented animals had higher enzyme activity (p = 0.004) in liver. Cattle with pigmented fat had higher expression and activity of betaCO in liver, but its level was not high enough to prevent the storage of beta-carotene in adipose tissues.