An RNA-dependent DNA polymerase associated with intracisternal A particles has been characterized. The enzyme required Mg(2+) or Mn(2+), dithiothreitol and the presence of all four deoxyribonucleoside triphosphates for the expression of maximal activity. Sensitivity of the endogenous RNA-dependent DNA polymerase activity to a low concentration of pancreatic ribonuclease in the presence of a high concentration of NaCl suggested that the enzyme might be utilizing the A particle endogenous RNA as template. Evidence in support of this was provided by analyses of early and late DNA products of the endogenous reaction by Cs(2)SO(4) isopycnic gradient centrifugation and hybridization of purified 60 to 70S and 35S RNAs of A particles with the purified DNA product.