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<p>We present new estimates of the regional North Atlantic (15–80<span class="inline-formula">^∘</span> N) CO<span class="inline-formula">₂</span> flux for the 2000–2017 period using atmospheric CO<span class="inline-formula">₂</span> measurements from the NOAA long-term surface site network in combination with an atmospheric carbon cycle data assimilation system (GEOS-Chem–LETKF, Local Ensemble Transform Kalman Filter). We assess the sensitivity of flux estimates to alternative ocean CO<span class="inline-formula">₂</span> prior flux distributions and to the specification of uncertainties associated with ocean fluxes. We present a new scheme to characterize uncertainty in ocean prior fluxes, derived from a set of eight surface <span class="inline-formula"><i>p</i></span>CO<span class="inline-formula">₂</span>-based ocean flux products, and which reflects uncertainties associated with measurement density and <span class="inline-formula"><i>p</i></span>CO<span class="inline-formula">₂</span>-interpolation methods. This scheme provides improved model performance in comparison to fixed prior uncertainty schemes, based on metrics of model–observation differences at the network of surface sites. Long-term average posterior flux estimates for the 2000–2017 period from our GEOS-Chem–LETKF analyses are <span class="inline-formula">−</span>0.255 <span class="inline-formula">±</span> 0.037 PgC yr<span class="inline-formula">⁻¹</span> for the subtropical basin (15–50<span class="inline-formula">^∘</span> N) and <span class="inline-formula">−</span>0.203 <span class="inline-formula">±</span> 0.037 PgC yr<span class="inline-formula">⁻¹</span> for the subpolar region (50–80<span class="inline-formula">^∘</span> N, eastern boundary at 20<span class="inline-formula">^∘</span> E). Our basin-scale estimates of interannual variability (IAV) are 0.036 <span class="inline-formula">±</span> 0.006 and 0.034 <span class="inline-formula">±</span> 0.009 PgC yr<span class="inline-formula">⁻¹</span> for subtropical and subpolar regions, respectively. We find statistically significant trends in carbon uptake for the subtropical and subpolar North Atlantic of <span class="inline-formula">−</span>0.064 <span class="inline-formula">±</span> 0.007 and <span class="inline-formula">−</span>0.063 <span class="inline-formula">±</span> 0.008 PgC yr<span class="inline-formula">⁻¹</span> decade<span class="inline-formula">⁻¹</span>; these trends are of comparable magnitude to estimates from surface ocean <span class="inline-formula"><i>p</i></span>CO<span class="inline-formula">₂</span>-based flux products, but they are larger, by a factor of 3–4, than trends estimated from global ocean biogeochemistry models.</p>