AbstractAlbumin bindingfor the enantiomers of four closely relatedN‐methyl‐5‐phenyl‐5‐alkyl‐barbiturates1–4was investigated for three different mammalian species by means of equilibrium dialysis. Lipid solubility (n‐heptane/phosphate buffer distribution coefficient) increased stepwise by a factor of 56 from1to4.Bovine serum albumin:The (S)‐(+)‐enantiomers of1–4were bound in a higher percentage than the (R)‐(−)‐enantiomers; lengthening of the aliphatic side‐chain increased the binding extent in both enantiomeric groups.Human serum albumin:Binding of (S)‐(+)‐1and (S)‐(+)‐4was higher than that of the (R)‐(−)‐enantiomers; with (S)‐(+)‐2and (S)‐(+)‐3it was much lower than that of the corresponding (R)‐(−)‐enantiomers. Lengthening of the aliphatic side chain increased the binding extent of the (S)‐(+)‐ as well as of the (R)‐(−)‐enantiomers, but with two exceptions: 1. The (S)‐(+)‐1binding exceeded that of the (S)‐(+)‐2by a factor of nearly two 2. The binding extent of (R)‐(−)‐4was not further increased in comparison to (R)‐(−)‐3.Rat serum albumin:(S)‐(+)‐1and (S)‐(+)‐2were bound in a lower percentage than the (R)‐(−)‐enantiomers, both3‐enantiomers showing an equal binding extent; (S)‐(+)‐4was bound to a slightly greater extent than the (R)‐(−)‐4.In the group of the (S)‐(+)‐enantiomers, the binding extent increased from1to4, whereas in that of the (R)‐(−)‐enantiomers only between1and4.Structural differences between the serum albumins of three mammalian species possibly cause the enantioselective binding pattern found for the enantiomers of1–4, and are r