Vero cell cultures are used in the quality control of Diphtheria vaccines: to estimate vaccine potency and to determine residual toxicity and reversion to toxicity. The impact of replacing foetal calf serum containing medium (SCM) by serum free media (SFM) on the sensitivity of Vero cells to Diphtheria Toxin was studied. Compared to SCM, SFM showed an eight-fold decrease in sensitivity to Diphtheria Toxin. This decrease was almost immediate, indicating that this phenomenon was not caused by a change in membrane structure or protein expression. We investigated the effect of SFM on Diphtheria Toxin in order to determine the cause of the decrease in sensitivity. Our results show that oligopeptides, which are often used in SFM as part of the replacement of foetal calf serum, are the most likely cause.