Using batch cultures, the 15N/14N (hereafter δ15N) of diatom-bound organic matter was measured and compared to the δ15N of total diatom biomass during the progressive consumption of a nitrate pool in four polar diatom species (Fragilariopsis cylindrus, Fragilariopsis kerguelensis, Pseudo-nitzschia seriata, and Thalassiosira nordenskioeldii) and one temperate species (Thalassiosira aestivalis). In general, the δ15N of the dissolved nitrate in seawater was greater than that of the biomass, which was greater than that of the diatom-bound N. Rayleigh-type relationships were observed, allowing for estimation of the isotope effect (ɛ) for each species, with a range from 1.0‰ to 14.0‰ across all species. For all cultured strains, the δ15N values of the diatom-bound (δ15NDB) fraction was lower than those of the total diatom biomass (δ15Nbiomass). The isotopic offset between the biomass and diatom-bound N (δ15NDBoffset = δ15Nbiomass − δ15NDB) was relatively constant along the growth curve for each individual species but varied among species, with a range of 1.9‰–11.2‰. Weak relationships were determined when ɛ and the δ15NDBoffset were compared to cellular size and surface area:volume ratio. More significantly, with the exception of Pseudo-nitzschia seriata, a strong positive relationship was found between ɛ and δ15NDBoffset. While the culture data indicate a positive δ15NDBoffset across all studied diatom species, surface sediment data suggest a negative δ15NDBoffset for sedimentary assemblages. This indicates that either (1) the growth conditions of our cultures had some effect on δ15NDBoffset or (2) a low-δ15N component of the N that we measure as diatom frustule–bound is lost during early diagenesis. Given documented assemblage changes, our culture data for relevant species do not suggest that the higher δ15NDB observed in the Antarctic during ice ages can be explained by species related changes in the sedimentary bulk-to-diatom-bound isotopic difference. Future work on the diatom-bound material in cultured diatoms grown under in situ nutrient conditions, analysis of sediment trap and net tow material, and frustule dissolution experiments will more completely assess this paleoproxy.