Previous work has demonstrated that substrate-induced alterations of tumor metabolism can be exploited to potentiate tumor response to cycle-specific chemotherapy (methotrexate, Adriamycin (doxorubicinl). This study was performed to investigate the biologic mechanism responsible for this phenomenon by determining the effect of short-term total parenteral nutrition (TPN) on tumor cytokinetics. Forty-two female Lewis/Wistar rats with subcutaneous mammary tumor implants (AC-33) underwent superior vena caval cannulation, and were randomized to receive either TPN or normal saline intravenously. Animals receiving TPN were killed at 2, 6, 12, 24, and 48 hours after initiating TPN; control animals given normal saline were killed at 0, 24, and 48 hours after randomization. At the time the animals were killed tumor cytokinetic analysis was performed by flow cytophotometry. The percentage of tumor cells in S-phase was significantly increased in animals after only 2 hours of TPN (55.5 ± 9.1%) compared with the control group (43.7 ± 7.7%) (P < 0.01). The ratio of sensitive/resistant tumor cells to S-phase-specific chemotherapy was effectively increased in animals receiving adjuvant TPN (1.31 ± 0.43) compared with control animals (0.80 ± 0.25) (P < 0.015). This alteration in tumor cytokinetics provides one explanation for the enhanced tumor response to cycle-specific chemotherapy previously observed with pulse TPN administration. Cancer 53:1409-1415, 1984.