Water samples taken at three depth layers from the offshore oligotrophic Cretan Sea were analyzed for ultraphytoplankton size fractionation using different methods: (a) sequential filtration on filters of pore size 5, 1 and 0.2 microm, (b) separate filtration using filters 5 and 0.2 microm as well as 1 and 0.2 microm and (c) direct filtration on 0.2 microm filters after staining of the samples with DAPI. Total abundance of photosynthetic organisms as well as the abundance of different groups such as flagellates and cyanobacteria measured by means of sizing after DAPI staining were significantly higher than those obtained by the other methods. This indicates that although there were no significant differences between the estimates provided by the separate and sequential filtration, both these methods underestimated total abundance by at least 25-50%. The estimates for the size fractions were also found to range from relatively imprecise to completely unreliable depending on the group and the size range. Although size fractionation through direct observation after staining largely depends on the expertise of the observer, this study suggests that it may provide more informative estimates than the other two methods. Although it is difficult to generalize the results of this study in a global context, the paper provides strong indications on the limitations of the sequential and separate methods for size fractionation of photosynthetic organisms and implies that their results are likely to be less accurate than is presently believed.