A small upstream open reading frame (uORF), located 14 nucleotides from the cap in the 5' transcript leader (5' TL) of the mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC), suppresses translation of the downstream cistron in normal T cells and T cell lines. In the present study, we examined the structural features of the 5' TL that overcome this suppressive influence in cells of nonlymphoid origin. Initiation at the downstream cistron in nonlymphoid cells is by a cap-dependent mechanism that requires ribosome scanning along the 5' TL and does not involve an internal ribosome entry site. Extending the uORF so that it overlapped the major cistron by 101 nucleotides had no effect on translation of the downstream cistron in either HeLa or Jurkat cells. When the distance between the uORF and the cap was extended to 47 nucleotides, using sequence previously found to be neutral, translation of the major cistron was inhibited 5-fold in HeLa cells and the mRNA was moved from polysomes to monosomes, a location identical to that of the wild type mRNA in Jurkat cells. Therefore, in contrast to T cells, initiation at the uORF seems to be relatively infrequent in non-lymphoid cells due to its proximity to the cap, allowing efficient translation of the downstream cistron.