The effect of human serum and CO 2 on the activity of roxithromycin and erythromycin was assessed. Protein binding of roxithromycin in serum from various animal sources, acid αl-glycoprotein and human albumin V was determined. There was a four- to eight-fold increase in MIC and MBC of roxithromycin in the presence of 70 and 100% human serum (minimum effect seen with erythromycin) and for both compounds there was a four- to eight-fold increase in MIC for fastidious organisms in the presence of CO 2 . Roxithromycin appears to be selectively bound to acid αl-glycoprotein, binding decreases with an increase in roxithromycin concentration (saturable at 10mg/l) and protein binding is variable depending on animal source (86% human, 10% guinea pig) and this must be considered when data on activity from animal studies are evaluated.