Electrophoretically homogeneous messenger RNA for rat preproinsulin has been prepared from an islet cell tumor by the use of oligo(dT(-cellulose chromatography and sucrose density gradient centrifugation. The molecular weight of the mRNA is about 210,000, as determined by polyacrylamide gel electrophoresis in formamide, and its sedimentation coefficient is 9.3 S in sucrose gradients containing 0.2 M NaCl. These results indicate that the synthesis of preproinsulin is directed by a monocistronic mRNA 600 nucleotides in length and requires approximately 55% of the molecule's coding capacity.