In bacteriophage T4, a major portion of DNA replication is initiated at random along the map, although several proven and putative origins have been described for early replication. In order to analyze the contribution of transcription and translation as well as DNA replication to intra-strand bias from A=T and G=C, we examined the pattern of the intra-strand biases in the first, second, and third codon positions of the coding regions as well as the intergenic regions of the T4 genome. We found, along the map, characteristic biases both from A=T and G=C for each codon position and the intergenic regions. The bias patterns were closely associated with the location of the sense and anti-sense segments in the genome. The results suggest that: (1) transcription-associated mutation is likely a significant cause of the bias, which is suggested by the pattern of the AT bias (bias from A=T) in the third codon position; (2) DNA replication coupled bias may also exist, which is suggested by the pattern of the GC bias (bias from G=C) in the third codon position and the intergenic regions; and (3) the bias patterns of the first and second codon positions of the sense segments are consistent with universal properties of the coding sequence that G is in excess and T is deficient in the first codon position, and Gis deficient in the second codon position. (C) 1999 Elsevier Science B.V. All rights reserved.