In large-scale mammalian cell culture, the key toxic by-products assessed and monitored are lactate and ammonia. Often no distinction between the two isoforms of lactate is made. Here, we present profiles of both D-and L-lactate. D-Lactate is the end molecule of the methylglyoxal pathway. D-Lactate unlike L-lactate is not re-utilized, and although during normal culture time frames it represents one-tenth of total lactate, during lactate re-use it represents nearly 35%. This indicates significant carbon flow through pathways not associated with primary metabolites. We have observed that the behavior of D-lactate is radically different from that of L-lactate with the level of one isoform changing, whilst the concentration of the other remains constant. This is an example of an alternate carbon flow pathway containing metabolic intermediates that may potentially have a detrimental effect on cells. The activity of the methylglyoxal pathway when measured as a proportion of glucose consumption in this study far exceeds any previously reported. This highlights the potential importance of "non-primary'' metabolisms to long lifespan mammalian fermentation practices. Biotechnol. Bioeng. 2010; 107: 182-189. (C) 2010 Wiley Periodicals, Inc.