In the course of measuring the concentrations of retinoic acids (RA) in bovine plasma, a major peak was observed which comigrated with 9-cis-RA on normal-phase high-performance liquid chromatography. Rechromatography of this retinoic on reverse-phase high-performance liquid chromatography showed that it was distinct from 9-cis-,13-cis-, and all-trans-RA, but comigrated with 9-cis,13-cis-retinoic acid (9,13-di-cis-RA). This retinoid was identified as 9,13-di-cis-RA based on its chemical, spectral, and chromatographic properties. Plasma concentrations of 9,13-di-cis-RA increased from < or = 0.5 ng/mL at birth to 5-6 ng/mL by 48 h of age in the calf. The 9,13-di-cis-RA was also a major circulating product of 9-cis-RA dosed intramuscularly to rats; conversely, intravenous administration of 9,13-di-cis-RA produced circulating 9-cis-RA in the rat. 9,13-Di-cis-RA had little or no affinity for cellular retinoic acid binding protein types I and II. This study establishes 9,13-di-cis-RA as a naturally-occurring retinoid under physiological conditions, shows that it undergoes interconversion with 9-cis-RA, and emphasizes a need for careful chromatography to resolve 9-cis-RA and 9,13-di-cis-RA. This is consistent with in vivo 13-cis isomerization operating to modify the concentration and perhaps the activity of 9-cis-RA in vivo.