Purification of casein messenger ribonucleic acids (mRNAs) from lactating rat mammary gland RNA has been accomplished by a combination of sizing techniques, including Sepharose 4B chromatography and preparative agarose-urea gel electrophoresis, and affinity chromatography of poly(adenylic acid)-containing mRNA on oligo(dT)-cellulose. The separation of the individual casein mRNAs into discrete molecular species free of apparent ribosomal RNA contaminants was facilitated by the use of denaturing conditions either prior to or during each of the fractionation procedures. Two casein mRNA fractions were isolated: (1) a 15S mRNA doublet which directed the synthesis of the two largest rat caseins in the wheat-germ, cell-free, translation assay, and (2) a 12S mRNA which migrated as a single species during agarose-urea gel electrophoresis and directed the in vitro synthesis of the smallest of three rat caseins. These mRNAs had apparent molecular weights of 450 000 +/- 30 000 and 320 000 +/- 25 000 and contained poly(adenylic acid) sequences at their 3' termini ranging from 15 to 150 residues with number average lengths of 42 and 38 adenosines, respectively. The purity of the isolated casein mRNA'S was determined both by agarose-urea gel electrophoresis and by a careful comparison of the total products synthesized in the wheat-germ translation assay with those recognized by a specific casein antibody using an indirect immunoprecipitation technique. The specificity of the indirect immunoassay procedure was demonstrated by the selective displacement by purified rat casein of greater than 95% of the radioactive product synthesized in the cell-free system. Under optimal translation conditions for casein mRNA, at least 90% of the released protein synthesized in response to the 15S casein mRNA was specifically immunoprecipitable, representing a 178-fold purification compared with the initial RNA extract. Using these techniques a comparable purification was also obtained for a 15S mouse casein mRNA fraction. Finally, an analysis by fluorography on 5-20% (w/v) polyacrylamide gradient slab gels of the total proteins synthesized in response to both the 15S and 12S casein mRNAs revealed a close correspondence with those proteins which were specifically immunoprecipitated.