Rhinovirus type 14 (RV14) incuced a transient statistically significant stimulation in synthesis of DNA which appeared between 0 and 3 h post-inoculation in the cytoplasm of high density monolayer cultures of KB cells. Newly synthesized DNA was measured by incorporation of [3H] thymidine into acid-insoluble DNAase-sensitive material and the cytoplasmic location established by cell fractionation and electron microscope radioautographic methods. A minimum of 10 plaque-forming units per cell of RV14 was required to stimulate DNA synthesis which did not occur above 34.5 degrees C, a temperature optimal for virus replication. Cytoplasmic DNA taken from RV14-infected or control cells could be differentiated from the bulk of cell (nuclear) DNA by several criteria, including: (1) RV14 induction of synthesis; (2) lower buoyant density and greater heterogeneity in CsCl and ethidium bromide/CsCl gradients; and (3) a different kinetic complexity upon reannealing. The Cot 1/2 value of cytoplasmic DNA, calclated as 50--100 from reassociation profiles, was about 10-fold less complex than the Cot 1/2 value of nuclear DNA (800-1000). These data rule out the possibility that cytoplasmic DNA arises by random breakage of nuclear DNA during cell disruption and extraction and are compatible with the hypothesis that inoculation of KB cells with RV14 results in stimulation of synthesis of a specific class of cell DNA which is detected in the cytoplasm.