The disaccharidases are important digestive enzymes, whose activities can be reduced by iron deficiency. We hypothesise that this is due to reduced gene expression; either by impairment to enterocyte differentiation, or by iron-sensitive mechanisms that regulate mRNA levels in enterocytes. Iron deficient Wistar rats were generated by dietary means. The enzyme activities and kinetics of sucrase and lactase were tested, as well as the activity of alkaline phosphatase (IAP-II) since it is unrelated to carbohydrate digestion. mRNA levels of -actin, sucrase, lactase, and the associated transcription factors PDX-1, CDX-2, GATA-4 and HNF-1 were measured by real-time PCR. Spatial patterns of protein and gene expression were assessed by immunofluorescence and in situ hybridisation respectively. It was found that iron deficient rats had significantly lower sucrase (19.5% lower) and lactase (56.8% lower), but not IAP-II activity than control rats. Kinetic properties of both enzymes remained unchanged from controls, suggesting a decrease in the quantity of enzyme present. Sucrase and lactase mRNA levels were reduced by 44.5% and 67.9% respectively by iron deficiency, suggesting enzyme activity is controlled primarily by gene expression. Iron deficiency did not affect the pattern of protein and gene expression along the crypt to villus axis. Expression of PDX-1, a repressor of sucrase and lactase promoters, was 4.5 fold higher in iron deficiency while CDX-2, GATA-4 and HNF-1 levels were not significantly different. The data suggest that decreases in sucrase and lactase activities result from reduction in gene expression, following from increased levels of the transcriptional repressor PDX-1.