Excess accumulation of lipids in nonadipose tissues such as skeletal muscle and liver has been implicated in the development of obesity‐related disorders, but the cause of this ectopic lipid overload remains unknown. The aim of this study was to determine in vivo postprandial lipid partitioning in rat skeletal muscle and liver, using localized ¹H‐[¹³C] magnetic resonance spectroscopy in combination with the oral administration of ¹³C‐labeled lipids. Six rats were measured at baseline and 5 and 24 h after administration of 400 mg [U‐¹³C]‐labeled algal lipids. Five hours after administration, fractional ¹³C enrichments of the lipid pools in muscle and liver were increased 3.9‐fold and 4.6‐fold (P < 0.05), respectively, indicating that part of the ingested lipids had been taken up by muscle and liver tissue. At 24 h, fractional ¹³C enrichments of muscle and liver lipids were decreased 1.6‐fold and 2.2‐fold (P < 0.05), respectively, compared with the 5 h values. This can be interpreted as a depletion of ¹³C‐labeled lipids from the intracellular lipid pools as a consequence of lipid turnover. In conclusion, the novel application of ¹H‐[¹³C] magnetic resonance spectroscopy in combination with the oral administration of ¹³C‐labeled lipids is applicable for the longitudinal assessment of in vivo lipid partitioning between multiple tissues. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.