2-Butoxyethanol is a glycol ether widely used in printing inks, varnishes and cleaning fluids. As skin absorption can be significant, biological monitoring is useful in monitoring worker exposure. A number of analytes and matrices have been used previously, including 2-butoxyethanol in blood and free and total 2-butoxyacetic acid in urine. Using a combination of a volunteer study and samples from exposed workers, we compared the applicability of some of the biological monitoring markers available. We conclude that 2-butoxyethanol in blood is not a suitable marker for biological monitoring due to sampling problems. In view of the low-level exposures reported in occupational surveys, 2-butoxyethanol in breath is also unsuitable because of a lack of sensitivity. Measuring 2-butoxyacetic acid in blood is possible, although non-invasive urine samples are preferred. Free 2-butoxyacetic acid in urine has previously been widely used; however, we found that the extent of conjugation of 2-butoxyacetic acid in urine varied from 0 to 100% both within and between individuals and is not related to time, concentration or urine pH. Data from 48 exposed workers suggested that an estimated 57% (95% confidence interval 44-70%) of the total 2-butoxyacetic acid is excreted in the conjugated form, and that conjugation may be activated above a certain exposure level. Using total 2-butoxyacetic acid significantly reduced inter-individual variation. Elimination half-lives for free and total 2-butoxyacetic acid were similar ( approximately 6 h) and there was no delay in excretion of the conjugated metabolite (peak excretion for both free and total was between 6 and 12 h after the end of exposure). In conclusion, we propose that total butoxyacetic acid (after acid hydrolysis) in urine is the biomarker of choice for monitoring exposure to 2-butoxyethanol. Urine samples should be collected post-shift towards the end of the working week.