Metabolic fates of fatty acids (FA) may be influenced by circulating FA concentration. Previous work in our lab demonstrated an ability of C18:3n-3 cis to ameliorate gene expression of pyruvate carboxylase (PC) after depression by either C16:0 or C18:0. PC catalyzes oxaloacetate (OAA) synthesis and ostensibly links gluconeogenesis and FA metabolism. Our objective was to determine effects of copresence of saturated and unsaturated FA pretreatments on cellular partitioning of [1-¹⁴C] C16:0 metabolism to CO₂ or acid-soluble products (ASP) in Madin-Darby bovine kidney (MDBK) cells. Cells at 80% confluence were exposed for 21h to either individual FA bound to BSA (C16:0, C18:0, C18:1n-9 cis or C18:3n-3 cis) or FA cocktails in 10:90, 25:75, 50:50, 75:25 or 90:10 ratios for combinations of C16:0: C18:3n-3 cis or C18:0: C18:3n-3 cis or C18:1n-9 cis: C18:3n-3 cis. Total pretreatment FA concentration was 1.0 mM. Following pretreatment, cells were then incubated in the presence of 1.0 mM [1-¹⁴C] C16:0 for 3h. Pretreatments with either C16:0 alone or C18:0 alone significantly (P < 0.01) depressed subsequent oxidation of [1-¹⁴C] C16:0 to ASP by 62.7% and 41.2%, respectively, compared with C18:3n-3 cis pretreatments. Pretreatments with C18:1n-9 cis either alone or in any combination with C18:3n-3 cis did not significantly (P > 0.10) depress subsequent oxidation of [1-¹⁴C] C16:0 to ASP. Similar patterns were seen with [1-¹⁴C] C16:0 oxidation to CO₂. ASP production from [1-¹⁴C] C16:0 was positively correlated (r = 0.68, P < 0.01) with PC gene expression levels while CO₂ production from [1-¹⁴C] C16:0 did not show a correlation (r = 0.30, P > 0.10) with PC expression. Activation of PC gene expression by unsaturated FA may play a critical role in setting the capacity for OAA synthesis and determining metabolic fates of FA. Results show the regulation of ketone production by bovine kidney cells in response to saturated and unsaturated FA pretreatments.