After translation of total rat intestinal RNA, immunoprecipitation using monospecific antiserum against rat intestinal alkaline phosphatase yielded two polypeptides in the adult duodenum and jejunum (molecular masses 62 and 65 kDa). Immunoprecipitation of both bands was blocked by a single purified alkaline phosphatase. In the adult ileum and in the entire small intestine of suckling pups, only the 62 kDa translation product was found. After fat feeding, translated alkaline phosphatase increased by an amount proportionate to the increase in enzyme activity previously seen in the serum. A small fraction of nascent alkaline phosphatase was translocated into microsomal vesicles, producing peptides of 65 and 69 kDa. Tunicamycin-treated membranes demonstrated a different signal peptide for each translation product. N-Terminal sequencing of the translation products showed leucine residues at similar positions, but overlap with the mature protein sequence was not demonstrated. On the basis of these data, we propose the presence of two mRNAs encoding alkaline phosphatase in the rat intestine.