The extents of protein binding of an N-pyrrolyl derivative penicillin in plasma of different species were determined in vitro by the equilibrium dialysis technique and spectrophotometry determination. The percentage of binding was determined in cows, sheep, pigs and dogs. The percentage of drug that was protein bound was independent of drug concentration for this penicillin within the range studied (5 to 40 micrograms ml-1). The extent of binding was determined at 20 micrograms ml-1. There was a significant difference in the extent of penicillin binding between species (t test P less than 0.005) except for between cows and dogs (t test P less than 0.05) and sheep and pigs (where it was not significantly different). In the species studied the extent of penicillin binding ranged from 41 to 55 per cent.