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 co-presence 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. We hypothesized that the ratio of saturated to unsaturated FA pretreatments regulates [1-¹⁴C] C16:0 partitioning to CO₂ or ASP. 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 and was applied in triplicate to 3 cell replicates. Following pretreatment, cells were incubated in the presence of 1.0 mM [1-¹⁴C] C16:0 for 3h before CO₂ and ASP collection. Data were analyzed using PROC MIXED of SAS. The model accounted for fixed effects of pretreatment and random effects of replicate. Pretreatments with either C16:0 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. Similar patterns were seen with [1-¹⁴C] C16:0 oxidation to CO₂. ASP production from [1-¹⁴C] C16:0 positively correlated (r = 0.68, P < 0.01) with PC gene expression from previous experiments. CO₂ production from [1-¹⁴C] C16:0 did not correlate (r = 0.30, P > 0.10) with PC expression. In conclusion, activation of PC gene expression by C18:3n-3 cis may play a critical role in setting the capacity for OAA synthesis and determining metabolic fates of FA. Results show a regulation of ketone production by MDBK cells in response to saturated and unsaturated FA pretreatments.