Background and Aims: Rats produce two messenger RNA (mRNA) isoforms encoding intestinal alkaline phosphatase that are regulated differentially by fat feeding but whose tissue distribution is not known. The aim of this study was to examine the distribution of these isoforms along the crypt/villus axis during fat feeding. Methods: Nucleotide probes and polyclonal antibodies unique for each isoform were used for in situ hybridization and immunocytochemistry. Results: mRNAs encoding isoforms I and II were distributed along the entire villus. The mRNA content of isoform II was more abundant. Quantitative grain counts showed that mRNA abundance of each isoform in the upper third of the villus was significantly (40%-65%) lower than in the remaining villus in the fasting duodenum. After fat feeding, the mRNA content of both isoforms increased over the villus. Five hours after feeding, the content of the mRNA encoding only isoform II was increased in the upper third of the villus relative to the rest of the villus. Only the mRNA encoding isoform II was found in the crypt cells. However, no immunoreactive enzyme was present in the crypt cells. Conclusions: The spatial expression of the two rat intestinal alkaline phosphatase isoforms is regulated differently in response to fat feeding.