The temperature dependent fluorescence of Rhodamine B was used to investigate the temperature effect of several system parameters in a microfluidic chip. This was combined with computational fluid dynamics calculations. Limited air movement over the chip had no significant effect on the temperature of the fluid running through the chip. Also, fluid flow through the channels at 0.01 -8 mu l min(-1) had no effect on the chip temperature or heating and cooling dynamics. The temperature varied greatly over the length of the chip. During transient operation of the chip, the heat up and cool down rates varied over the chip, and were dependent on the distance to the heater. The thermal time constant for heat up was four to five times lower than for cool down. The results can be used as tools for operating a temperature controlled microfluidic chip. (c) 2008 Elsevier Ltd. All rights reserved.