α-Retinol (αR) is a structural isomer of retinol [vitamin A (VA)] that does not bind to serum retinol-binding protein (RBP). In this study, α-retinyl acetate (αRA) was synthesized and given orally (35 μmol) to VA-deficient lactating sows (n = 11) to assess its potential to trace RBP-independent retinol transport and tissue uptake. The αRA dose primarily appeared in sow serum as 4 α-retinyl esters (αRE) with peak serum total αR concentrations (the sum of the alcohol and ester forms) detected at 2 h (70 ± 23 nmol/L, mean ± SEM) postdose. From 0 to 40 h postdose, the percentage of serum total αR in the alcohol form did not increase. Rapid αR uptake into sow milk was observed with peak concentrations (371 ± 83 nmol/L) at 7.5 h postdose, consistent with the uptake of αRE from chylomicra. A high percentage of the αRA dose (62 ± 15%, mean ± SD) was present in the livers of sows (n = 6) killed 22-28 d postdose. Approximately 15-26% of the sow αRA dose was transferred to the livers of the nursing piglets (n = 17) after 3 d. In piglets and sows, a similar percentage of hepatic total αR was detected in the ester form as that of hepatic total retinol. Taken together, these data suggest that an oral dose of αRA effectively traces the uptake, esterification, chylomicron transport, and hepatic storage of retinol and may be useful for deciphering the role of RBP-independent delivery of retinol to other tissues.