Quantifying isoprene emissions using satellite observations of the formaldehyde (HCHO) columns is subject to errors involving the column retrieval and the assumed relationship between HCHO columns and isoprene emissions, taken here from the GEOS-CHEM chemical transport model. Here we use a 6-year (1996–2001) HCHO column data set from the Global Ozone Monitoring Experiment (GOME) satellite instrument to (1) quantify these errors, (2) evaluate GOME-derived isoprene emissions with in situ flux measurements and a process-based emission inventory (Model of Emissions of Gases and Aerosols from Nature, MEGAN), and (3) investigate the factors driving the seasonal and interannual variability of North American isoprene emissions. The error in the GOME HCHO column retrieval is estimated to be 40%. We use the Master Chemical Mechanism (MCM) to quantify the time-dependent HCHO production from isoprene, α- and β-pinenes, and methylbutenol and show that only emissions of isoprene are detectable by GOME. The time-dependent HCHO yield from isoprene oxidation calculated by MCM is 20–30% larger than in GEOS-CHEM. GOME-derived isoprene fluxes track the observed seasonal variation of in situ measurements at a Michigan forest site with a −30% bias. The seasonal variation of North American isoprene emissions during 2001 inferred from GOME is similar to MEGAN, with GOME emissions typically 25% higher (lower) at the beginning (end) of the growing season. GOME and MEGAN both show a maximum over the southeastern United States, but they differ in the precise location. The observed interannual variability of this maximum is 20–30%, depending on month. The MEGAN isoprene emission dependence on surface air temperature explains 75% of the month-to-month variability in GOME-derived isoprene emissions over the southeastern United States during May–September 1996–2001.