Wheat yield and grain nitrogen concentration (GNC; mg N/g grain) are frequently negatively correlated. In most growing conditions, this is mainly due to a feedback process between GNC and the number of grains/m2. In Mediterranean conditions, breeders may have produced cultivars with conservative grain set. The present study aimed at clarifying the main physiological determinants of grain nitrogen accumulation (GNA) in Mediterranean wheat and to analyse how breeding has affected them. Five field experiments were carried out in north-eastern Spain in the 2005/06 and 2006/ 07 growing seasons with three cultivars released at different times and an advanced line. Depending on the experiment, source-sink ratios during grain filling were altered by reducing grain number/m2 either through pre-anthesis shading (unshaded control or 0.75 shading only between jointing and anthesis) or by directly trimming the spikes after anthesis and before the onset of the effective grain filling period (un-trimmed control or spikes halved 7–10 days after anthesis). Grain nitrogen content (GN content ; mg N/grain) decreased with the year of release of the genotypes. As the number of grains/m2 was also increased by breeding there was a clear dilution effect on the amount of nitrogen allocated to each grain. However, the increase in GN content in old genotypes did not compensate for the loss in grain nitrogen yield (GNY) due to the lower number of grains/m2. GN content of all genotypes increased (increases ranged from 0.13 to 0.40 mg N/grain, depending on experiment and genotype) in response to the post-anthesis spike trimming or pre-anthesis shading. The degree of source-limitation for GNA increased with the year of release of the genotypes (and thus with increases in grain number/m2) from 0.22 (mean of the four manipulative experiments) in the oldest cultivar to 0.51 (mean of the four manipulative experiments) in the most modern line. It was found that final GN content depended strongly on the source-sink ratio established at anthesis between the number of grains set and the amount of nitrogen absorbed at this stage. Thus, Mediterranean wheat breeding that improved yield through increases in grain number/m2 reduced the GN content by diluting a rather limited source of nitrogen into more grains. This dilution effect produced by breeding was further confirmed by the reversal effect produced by grain number/m2 reductions due to either pre-anthesis shading or post-anthesis spike trimming.