To test if incorporation profile of branched-chain VFA (BCVFA) or AA (BCAA) influenced elongation into bacterial FA, we dosed ¹³C-labeled BCVFA and BCAA into batch cultures of mixed rumen microbes. Treatments were 1) Control, 2) ¹³C-enriched isovalerate, isobutyrate, 2-methylbutyrate, and valerate (1 mM final concentration each), 3) ¹³C-enriched Ile, Leu, and Val (1 mM final concentration each), or 4) a 50:50 mix of BCVFA and BCAA (MIX). In 2 replicate tubes, 30 mL of a 1:4 dilution of blended rumen fluid in artificial saliva buffer was added anaerobically to 50-mL tubes with either 0.25 g of corn + 0.25 g of alfalfa hay (AHC) or 0.25 g of corn + 0.25 g of orchardgrass hay (OHC). Total FA in bacteria harvested at 24 h of incubation was not affected by treatment or treatment x feed interaction (P > 0.55). Adding BCVFA or BCAA increased (P < 0.05) 13:0, iso 14:0, and total odd chain FA (OCFA; g/ 100 g total FA). Total dose recovered in FA was not affected (P > 0.15) by BCAA (0.169%), MIX (0.193%), or BCVFA (0.206%). Linear (L) and quadratic (Q) contrasts were evaluated with PROC MIXED in SAS (2 runs as random effect) for 1) BCAA, 2) MIX, and 3) BCVFA. For dose recovery in 13:0, iso 16:0, 17:0, and iso 17:0, there was a L increase (P < 0.05) in enrichment for BCVFA substitution over BCAA (Q was P > 0.15). There was a treatment x feed interaction (P < 0.02) for anteiso 14:0, 15:0, anteiso 17:0, and OCFA enrichment. With AHC, anteiso 14:0 increased enrichment linearly (P < 0.03) at a decreasing rate (P = 0.12 for Q) with BCVFA substituting for BCAA. For 15:0 and total OCFA, L and Q were both P < 0.01, with enrichment increasing linearly at a decreasing rate with BCVFA substituting for BCAA. With OHC, 15:0, anteiso 17:0, and total OCFA enrichment increased linearly (P < 0.03; P > 0.15 for Q) as BCVFA replaced BCAA. Enrichment of anteiso 14:0 was highest with MIX (P > 0.15 for L; P < 0.01 for Q). The bacterial FA profile was similar among treatments, but the enrichment of the OCFA and several BCFA was greatly increased by dosing BCVFA over dosing BCAA, or a MIX, supporting a potential benefit from higher concentration of elongation primers regardless of substrate.