The tearing and burning sensations associated with raw onion consumption are caused by (Z,E)-propanethial S-oxide, the lachrymatory factor (LF). The LF is produced from the hydrolysis of S-1-propenyl-L-cysteine S-oxide (PREN), the dominant flavor compound in onions. Current methodology for LF quantification was optimized for Granex-type onions using a 2-min incubation time to allow for maximum formation. In this study, data were taken on PREN hydrolysis of `Dehydrator #3' and `Granex 33' at harvest and during storage and were compared to LF formation. `Dehydrator #3' PREN hydrolysis was 98% complete 5 s after cellular disruption at each sampling date. However, using the 2-min incubation procedure, only 10.25 μmol of LF was recovered from the hydrolysis of 30.11 μmol of PREN at harvest, thereby underestimating LF for this cultivar. Percent PREN hydrolysis for `Granex 33' was lower than `Dehydrator #3' during the enzymatic reaction at each sampling date, suggesting slower PREN hydrolysis activity. At harvest, 6.96 μmol of LF were recovered from 12.54 μmol of PREN hydrolyzed. After 2 storage months, however, micromol of LF were equal to micromol of PREN. LF quantification is currently being considered by the onion industry as a direct measurement of gross onion pungency. This data suggests that more optimization of LF quantification is needed before it can be applied to a broad range of cultivar types.