Infant botulism is a rare disease in the UK, with the first case being recognized in 1978 and only five subsequent cases being reported before 2007. This study reports two unlinked cases of infant botulism, caused by two distinct strains of Clostridium botulinum (toxin types A and B, respectively), that occurred within a single month in the south-east of England in October 2007. The use of real-time PCR to detect C. botulinum neurotoxin genes in clinical specimens to improve the diagnostic procedure and to follow carriage of the causative organism in the infant gut is described. The laboratory investigation of these two cases demonstrated that a combination of the mouse bioassay, real-time PCR assays and conventional microbiological culture can provide rapid confirmation of a clinical diagnosis and affect patient management. Both infants (aged 4 and 8 months) were previously healthy prior to the onset of symptoms, and in both cases, a diagnosis of infant botulism was delayed for at least 10 days after initial admission to hospital. Once diagnosed, one of the infants was the first in the UK to be treated with human-derived botulism immunoglobulin. Real-time PCR was used to demonstrate that C. botulinum was excreted in the infants' faeces for up to 68 and 81 days, respectively. Despite the infrequency of infant botulism in the UK, clinicians should be aware of this rare but serious condition and should seek microbiological advice when presented with young infants with compatible symptomologies.