The nucleotide sequence of the segment of simian virus 40 DNA between standard map positions 0.53 and 0.65, i.e., approximately half of the restriction fragment Hind A, is reported. This segment is located near the beginning of the early region and is transcribed counterclockwise. There is a potential initiating ATG signal at 13 nucleotides from the Hind C-Hind A junction in the strand with the same polarity as the early mRNA. From this signal on, an open reading frame is present which would allow the synthesis of a polypeptide of 174 amino acids until a TAA termination codon is reached at nucleotide 602 (map position 0.547). This polypeptide, revealed by the DNA sequence, corresponds almost certainly to small-t antigen. Correlation of the deduced amino acid sequence with the NH(2)-terminal sequences of small-t and large-T (tumor) antigens of simian virus 40, as established by Paucha et al. [Paucha, E., Mellor, A., Harvey, R., Smith, A. E., Hewick, R. M. & Waterfield, M. D. (1978) [Proc. Natl. Acad. Sci. USA 75, 2165-2169], strongly argues that both proteins are indeed initiated at the ATG triplet. Because the DNA region between 0.547 and 0.534 is blocked for translation in all three reading frames by multiple termination condons, we conclude that the large-T antigen must be coded for by two noncontiguous DNA segments: the segment from 0.65 to around 0.60, which small-t and large-T antigens share, and another segment starting at some point after position 0.534 and continuing counterclockwise until it terminates at map position 0.174. Small-t antigen is methionine-rich and has a remarkably high number of cysteine residues clustered mainly in its COOH-terminal half. It is rich in both basic and acidic residues, the former being slightly in excess.