The acute-phase response is a catabolic event involving increased waste of amino-nitrogen (N) via hepatic urea synthesis, despite an increased need for amino-N incorporation into acute-phase proteins. This study aimed to clarify the regulation of N elimination via urea during different phases of the tumor necrosis factor-α (TNF-α)-induced acute-phase response in rats. We used four methods to study the regulation of urea synthesis: We examined urea cycle enzyme mRNA levels in liver tissue, the hepatocyte urea cycle enzyme proteins, the in vivo capacity of urea-N synthesis (CUNS), and known humoral regulators of CUNS at 1, 3, 24, and 72 h after TNF-α injection (25 μg/kg iv rrTNF-α) in rats. Serum acute-phase proteins and their liver mRNA levels were also measured. The urea cycle enzyme mRNA levels acutely decreased and then gradually normalized, whereas the urea cycle enzyme proteins remained essentially unchanged over time. The CUNS rose after 3 h and then normalized. The acute-phase response was fully activated at 24 h with markedly increased serum levels of the acute-phase proteins. TNF-α acutely upregulated the CUNS. Later, despite the fully established 24-h acute-phase response and the decreased activity of the urea cycle enzyme genes, there was no change in the urea cycle enzyme proteins or the CUNS. Thus in no phase after the initiation of the acute-phase response was in vivo urea synthesis orchestrated in combination with acute-phase protein synthesis so as to limit N waste.