John M. Chirgwin,** Alan E. Przybyla, 5 Raymond J. MacDonald, 11 and William J. Rutter* abstract: Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenizationin a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercapto-ethanol to break protein disulfide bonds. The RNA was iso-Ahe preparation of undegraded ribonucleic acid from a num-ber of cell types is hindered by the presence of active nucleases. An extreme example of this is the rat pancreas which contains over 200 pg of ribonuclease A per g of tissue wet weight (Beintema et al., 1973). Within the pancreatic exocrine cells, ribonuclease A as well as other digestive enzymes and zymo-gens appears to be synthesized on ribosomes bound to the cytoplasmic face of the endoplasmic reticulum, extruded di-rectly into the cisternal side, and subsequently packaged in secretory granules. Thus, the functions of the cytosol are effectively sequestered from these strong hydrolytic activities. Disruption of the cells, however, inevitably results in rapid mixing of RNA and RNase. 1'2* One way to eliminate nu-cleolytic degradation ofRNA is to denature allof the cellular proteins including RNase. This approach would be successful only if the rate of denaturation exceeds the rate of RNA hydrolysis by RNase. Deproteinization procedures using guanidine hydrochloride (Cox, 1968) or phenol even in the presence of RNase inhibitors such as heparin, iodoacetate, and detergent (Parish, 1972) are insufficiently effective to yield intact RNA from the pancreas.