The current model for processing DNA during entry in the transformation of Streptococcus pneumoniae is that following double-strand cleavage of DNA bound at the cell surface, uptake of one strand proceeds linearly from a newly formed 3'-end with simultaneous degradation of the opposite strand. Two important predictions of this model have been tested in the work reported here: first that the polarity of DNA degradation is the opposite of that for entry, and second that the rate of DNA degradation is (at least) equal to the rate of entry. The processing of DNA during entry was investigated using donor molecules constructed in vitro and labeled in one strand only. With uniformly labeled donor molecules, an amount of label equivalent to that taken up by the cells was recovered in acid-soluble form in the transformation medium. Experiments with 3'- or 5'-end-labeled molecules revealed that whereas essentially all of the 3'-end label was susceptible to degradation, most 5'-end label was resistant. Kinetic analysis of both entry and degradation revealed very similar rates for these processes, about 100 nucleotides s-1 at 31 degrees C, suggesting that they occur concomitantly. Entry and degradation appear to proceed with opposite polarity, 3'-->5' for entry and 5'-->3' for degradation. A prediction of the entry model, that a single-strand interruption would inhibit the uptake of DNA sequences located 5' to the nick, was confirmed experimentally. Therefore, we suggest that an intact sugar phosphate backbone is required by the entry machinery for continuous uptake.