Six lignocellulosic hydrolysates produced through acid pretreatment were analyzed for the occurrence of formaldehyde, acetaldehyde, and glycolaldehyde. Acetaldehyde was found in all six (0.3-1.6 mM) and formaldehyde in four (≤ 4.4 mM), whereas glycolaldehyde was not detected. To assess the relevance of these findings, fermentations with yeast and formaldehyde or acetaldehyde were performed in the concentration interval 0.5-10 mM. Formaldehyde already inhibited at 1.0 mM, whereas 5.0 mM acetaldehyde was needed to obtain a clear inhibitory effect. After 24 h of fermentation, 1.5 mM formaldehyde reduced the glucose consumption by 85%, the balanced ethanol yield by 92%, and the volumetric productivity by 91%. The results show that formaldehyde and acetaldehyde are prevalent in pretreated lignocellulose and that formaldehyde in some cases could explain a large part of the inhibitory effects on yeast by lignocellulosic hydrolysates, as three of six hydrolysates contained ≥ 1.9 mM formaldehyde, which was shown to be strongly inhibitory.