Insulins acylated with fatty acids at the epsilon-amino group of LysB29 constitute a new class of insulin analogs, which are prolonged-acting due to albumin binding. In the present study it is shown that the affinity of fatty acid acylated insulins for albumin varies considerably (> 50-fold) among species. The relative affinities of acylated insulin for albumin in human, pig, and rabbit serum are about 1:1:5:35. The several fold higher binding affinity in rabbit serum than in pig serum is reflected in a relatively more protracted effect after sc injection in rabbits than in pigs. Due to the similar binding affinities in pig serum and human serum, the pig model should provide a useful estimate of the degree of protraction of acylated insulin in humans. The results emphasize that species differences in ligand binding can be of major importance in the preclinical evaluation of highly albumin bound drugs.