Phosphoketolase is a well characterised enzyme in Bifidobacterium species. However, limited information about this enzyme is available in higher organisms. Physiological characterisation of the filamentous fungus Penicillium chrysogenum, using ¹³C-labelling analysis, revealed that an unrecognised pathway was involved in formation of cytosolic acetyl-CoA. Comparison of the labelling pattern of pyruvate and acetyl-CoA showed that the ¹³C-content of pyruvate was higher than the corresponding labelling of acetyl-CoA, indicating that a less labelled source was contributing to the acetyl-CoA pool. Examination of previously published ¹³C data from Aspergillus nidulans showed the same trend. It was speculated that phosphoketolase activity could be contributing to the formation of acetyl-CoA in these fungi. Since the traditional methods for measuring phosphoketolase activity are tedious, two new enzyme assays were developed and used for identification of phosphoketolase activity. Furthermore, using genomic sequencing information, targets for creating an entire pathway from xylulose 5-phosphate to acetate was identified in A. nidulans.