Rat sera, 10-30 h after unilateral nephrectomy (UNI), enhance 3H-thymidine ("3H-Tdr) incorporation into DNA of incubating renal tissue from control rats. Stimulation is even greater when extracts from remaining growing kidneys 20 h after UNI are combined with sera from rats after UNI. UNI extracts, i.e., extracts from the kidney remaining after uninephrectomy, are nonstimulatory alone. UNI sera and UNI sera plus UNI extracts could theoretically augment 3H-Tdr incorporation into renal DNA via dilutional means rather than enhanced DNA synthesis. To determine if our results were secondary to enhanced DNA synthesis, we performed our in vitro assay using the labelling of nuclei via autoradiography as another index. The addition of UNI sera compared to sera from sham-operated rats (SHAM) in seven paired experiments enhanced incorporation of 3H-Tdr into DNA by 30% (p less than 0.02) and the addition of both UNI sera and UNI extracts compared to SHAM sera and SHAM extracts enhanced incorporation by 48% (p less than 0.001). Unlike a dilutional effect, nuclear labelling also increased in these same seven experiments: UNI sera versus SHAM sera increased 25% (p less than 0.05) and UNI sera + UNI extracts versus SHAM sera + SHAM extracts increased 37% (p less than 0.01). We conclude that UNI sera and UNI sera + UNI extracts enhance 3H-Tdr incorporation into DNA by augmenting DNA synthesis, driving cells into the "S" phase. The use of 3H-Tdr incorporation into DNA in our assay does estimate DNA synthesis.