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Since 2006 increasing numbers of laying hen flocks with decreased production have been reported in the Netherlands. At necropsy, birds from affected flocks showed multifocal areas of necrosis in the duodenum. Histologically the duodenum had moderate to marked villus atrophy and fusion with crypt hyperplasia and a mixed inflammatory infiltrate within the lamina propria underlying focal areas of degenerative epithelium. Multifocally, free within the intestinal lumen and associated with epithelial necrosis, were marked numbers of large rod-shaped bacteria. Anaerobic culturing and subsequent toxin typing revealed, in 19 out of 73 affected birds, the presence of Clostridium perfringens strains, either type A or type C harbouring the atypical allele of cpb2 and netB. Eighteen out of these 19 birds carried C. perfringens strains capable of producing beta2 toxin in vitro and all of these birds harboured C. perfringens strains capable of producing NetB toxin in vitro. In contrast, specific pathogen free (SPF) birds lacked gross or histological lesions in their duodenum, and C. perfringens type C was isolated from four out of 15 SPF birds tested. One of these isolates harboured the consensus three allele of cpb2 that produced beta2 toxin in vitro. None of the C. perfringens isolates originating from SPF birds harboured netB. These findings might indicate that the NetB toxin produced by C. perfringens is associated with subclinical necrotic enteritis in layers, whereas the involvement of beta2 toxin in subclinical necrotic enteritis, if any, might be variant dependent.