Trypsin, chymotripsin and elastase are the pancreatic enzymes required for neonatal rat heart tissue disaggregation. We have previously developed a procedure for the isolation of a mixture of these three enzymes from commercial crude-trypsin samples. Toxic materials present in certain crude-trypsin samples are removed during purification. An evaluation of this mixture was conducted for its ability to disaggregate neonatal rat heart tissue for cell culture. Large numbers of cells were released with minimal cellular damage as determined by their ability to survive and function in culture. Rat lung and kidney tissue also were disaggregated successfully and cultured with this preparation. It is apparent that this enzyme preparation has a potential for disaggregating a wide variety of tissues.