1. DNA-directed DNA polymerase gamma was isolated from epithelial cells of the rabbit small intestine, and characterized. 2. The molecular weight of the enzyme, determined by Sephadex G-200 filtration was 105 000 +/- 15%. 3. The enzyme showed preference for poly(A) replication on poly(A)-poly(dT) or poly(A)-oligo(dT) 12-18 templates, as compared with activated DNA. Poly(C)-oligo(dG)12-18 replication was not observed. 4. The Michaelis constants for dTTP in replication of activated DNA and poly(A)-oligo(dT)12-18 were 3.3 and 2.0 micron, respectively.