Structural analysis of DNA from primary hepatocytes has been made by chromatography on benzoylated-DEAE-cellulose. DNA was labelled in vivo by incorporation of 3 H-TdR after partial hepatectomy 2 weeks prior to animals being used to establish hepatocyte cultures. Structural change in DNA associated with the transition to primary culture was assessed by comparison of data with that obtained when DNA was isolated from the intact organ immediately after death. In terms of this comparison, DNA from primary hepatocytes was bound more extensively to benzoylated-DEAE-cellulose in the presence of 1.0 M NaCl. Thus 32% of primary hepatocyte DNA was recovered in the formamide-eluted fraction after chromatography, a result which reflected a higher proportion of DNA containing single-stranded regions. This proportion of formamide-eluted DNA in primary hepatocytes could not be further reduced by extensive shearing and could not be affected by manipulation of the isolation procedure. Analysis of DNA isolated from hepatocyte cell lines revealed structural characteristics similar to DNA isolated from intact liver. The data imply that extensive structural damage to DNA is a consequence of the primary status of the hepatocytes examined.