In a previous paper, we showed that naturally occurring conjugated linoleic acid (CLA) from butter fat is metabolized in vivo to higher metabolites such as conjugated diene (CD) 18:3, CD 20:3 and CD 20:4, all the while retaining the conjugated diene structure. In this paper, we describe the detection of two more metabolites with characteristic conjugated diene UV spectra. HPLC retention times, UV and MS spectra identified the CLA metabolites as CD 16:2 and CD 16:3. The accumulation of CD 16:2 was significantly higher than that of CD 16:3 in all tissues examined. Tissue distributions of CD 16:2 and CD 16:3 were similar, with plasma and adipose tissue showing the highest levels, while kidney had the lowest and the liver an intermediate level. CD 16 fatty acids accounted for about 20% of the total CLA metabolites. The kidney, however, was an exception where CD 16 fatty acids accounted for only 11% of total metabolites. Analyses of liver lipid classes showed that CD 16:2 and CD 16:3 were preferentially incorporated into neutral lipids. This preferential incorporation was very similar to CLA as shown previously. We hypothesize that CD 16:2 and CD 16:3 may be derived from partial beta-oxidation of CLA and CD 20:4, respectively, even though we cannot rule out that CD 16:3 may also be derived from CD 18:3 and CD 20:3. Incubation of skin human fibroblasts from X-linked adrenoleukodystrophy (ALD) patients with c9,t11 CLA showed that CD 16:2 formation in ALD cells was about 50% lower than control cells. This result may tempt to hypothesize that, at least in part, CD 16:2 is beta-oxidized in peroxisomes.