The efficiency of RNA 3'-end formation of spleen necrosis virus (SNV) is determined by the distance between the cap site and the poly(A) site. When the distance between the cap site and the poly(A) site was shorter than 500 bases, only 3-9% of the RNA was polyadenylated at the SNV poly(A) site. However, when the distance between the cap site and the poly(A) site was 1400 bases or more, 70% of the total RNA was polyadenylated at the SNV poly(A) site. In contrast, the poly(A) signal sequences of the thymidine kinase (tk) and SV40 late genes functioned at high efficiency, even with a distance between the cap site and the poly(A) site that was short enough to inactivate the SNV poly(A) signal. Therefore, this distance-dependent inactivation of RNA 3'-end formation is specific for SNV sequences and perhaps for related retroviruses. This finding explains the difference between the 5' and 3' poly(A) sites in many retrovirus RNAs.