Phosphodiester oligonucleotides are rapidly degraded in spleen cell cultures. The present studies were conducted to determine whether thymidine released from degradation of such oligonucleotides could be reutilized and compete with [3H]thymidine incorporation, thereby causing nonspecific inhibition of "proliferation" assays. Our studies in mitogen-stimulated mouse spleen cells demonstrate that "control" oligonucleotides that contain thymidine can cause more than 90% inhibition of [3H]thymidine incorporation. This inhibitory effect was generally dependent on the location of the thymidine within the oligonucleotide: oligonucleotides that had 3'-terminal thymidine(s) caused more suppression than those in which thymidines were at the 5' end. All oligonucleotides caused a modest but variable inhibition of [3H]uridine incorporation. Furthermore, [3H]thymidine incorporation was partially inhibited even by oligonucleotides that did not contain thymidine. We propose that investigators who use [3H]thymidine incorporation assays to assess antisense effects do so with caution. It may be prudent to use control oligonucleotides with the same number and location of thymidine bases and to confirm [3H]thymidine incorporation assays with other measures of cell proliferation.