Ceramides may mediate saturated fat–induced insulin resistance, but there are no data comparing ceramide concentrations between human tissues. We therefore performed lipidomic analysis of human subcutaneous (SCfat) and intra‐abdominal (IAfat) adipose tissue, the liver, and serum in eight subjects. The liver contained (nmol/mg tissue) significantly more ceramides (1.5–3‐fold), sphingomyelins (7–8‐fold), phosphatidylethanolamines (10–11‐fold), lysophosphatidylcholines (7–12‐fold), less ether‐linked phosphatidylcholines (2–2.5‐fold) but similar amounts of diacylglycerols as compared to SCfat and IAfat. The amounts of ceramides and their synthetic precursors, such as palmitic (16:0) free fatty acids and sphingomyelins, differed considerably between the tissues. The liver contained proportionally more palmitic, stearic (18:0), and long polyunsaturated fatty acids than adipose tissues. Stearoyl‐CoA desaturase 1 (SCD1) activity reflected by serum, estimated from the 16:1/16:0‐ratio, was closely related to that in the liver (r = 0.86, P = 0.024) but not adipose tissues. This was also true for estimated elongase (18:1/16:1, r = 0.89, P = 0.01), and Δ5 (20:4/20:3, r = 0.89, P = 0.012) and Δ6 (18:3[n‐6]/18:2, r = 1.0, P < 0.001) desaturase activities. We conclude that the human liver contains higher concentrations of ceramides and saturated free fatty acids than either SCfat or IAfat.