AbstractThe dynamic behavior of the Ribulose Monophosphate‐typeMethylomonasL3 in continuous cultures was studied, using methanol pulses to induce fast transients in steady‐state cultures of single (methanol) and mixed (methanol plus formaldehyde) substrates. In several experiments, the methanol‐uptake rate (MUR) profiles displayed negative MUR values for a time period following the methanol pulse, and significant amounts of methanol disappeared immediately following the pulse. These phenomena suggested the accumulation of methanol in the cells upon pulsing, apparently due to an active transport system. Accordingly, and in order to estimate the potential of the transport system for methanol accumulation, accumulation profiles were calculated for several pulse experiments. The calculations are based on a methanol balance and experimentally determined values of the cell volume and the true transient biomass yields. It is calculated that methanol accumulates up to 200‐fold to very high intracellular concentrations. The accumulation is calculated to be much higher in single‐ (methanol) substrate cultures of low dilution rate than in cultures of high dilution rate or of mixed substrates. The specific growth rate immediately following the methanol pulse decreased in single‐substrate cultures and increased in mixed‐substrate ones. The biomass yield decreased after the methanol addition in all experiments; however, the drop was less severe in the mixed‐substrate experiments. It is also suggested that formaldehyde as a methanol cosubstrate may be an effective means of providing more stable biomass yields and growth rates in reactors with imperfect mixing, and of protecting the reactor against accidentally induced methan