In human KB cells productively infected with adenovirus type 12 (Ad12) the kinetics of synthesis of Ad12-specific RNA were investigated. Using 2 h labelling periods, striking differences were observed in the time course and the rate of synthesis of virus RNA comparing the nuclear, cytoplasmic and polysomal RNA fractions. A protracted onset of virus RNA synthesis was followed by an exponential increase between 15 and 40 h p.i.; reaching a maximum at 40 to 45 h p.i., the fraction of the newly synthesized Ad12-specific RNA remained constant up to 66 h in the nucleus, but decreased in the cytoplasm. Characteristic patterns were found in the size distribution of Ad12-specific messenger RNA synthesized early and late in the infection cycle. By using 10 min labelling periods, the size distribution and the degree of polyadenylation of the primary transcripts were determined. Early in infection, the size distribution of the virus-specific nuclear RNA resembled that of the mRNA, whereas late after infection most of the label was found in high mol. wt. RNA sedimenting between 30 and 55S. Eighty percent of the nuclear RNA was polyadenylated and belonged to the high mol. wt. RNA class. During the early phase of infection, approx. 20% of the virus genome was found to be transcribed symmetrically, and later after infection the entire Ad12 genome. Symmetrical (self-complementary) transcripts were preferentially drived from the terminal parts of Ad12 DNA.