Autophosphorylation of phytochrome A (phyA) and transphosphorylation of its reaction partners, phytochrome kinase substrate 1 (PKS1) in particular, might play important functions in signal transduction from phyA. It was shown that PKS1 and PKS2 physically interact with phyA and phyB in vitro, and that overexpression of PKS1 interferes with phytochrome signaling in vivo. Moreover, both pks1 and pks2 loss of function mutants are specifically defective for one branch of phyA signaling. We therefore used in vivo fluorescence spectroscopy to test whether mutations in pks1 and pks2 or overexpression of PKS1 (PKS1OX) have an effect on phyA and its subpopulations, phyA′ and phyA″. It was found that the emission spectra of phyA in all the Arabidopsis lines are similar. The phyA content in the single mutants pks1 and pks2, and also in PKS1OX, was 1.2–1.5 times higher than in the wild type, whereas the phyA′/phyA″ ratio remained practically unchanged (approx. 1.0). However, in the double mutant pks1pks2, the picture is reversed—the phyA concentration remained unchanged, while the phyA′/phyA″ ratio shifted dramatically towards phyA″ (0.3). This suggests that (i) the changes in PKS1 or PKS2 content may affect the total phyA concentration, (ii) PKS1, together with PKS2, could be critical for the formation of phyA′, thus shifting the equilibrium towards phyA″ in the double mutant and (iii) these variations in the phyA′ and phyA″ content may contribute to the mutant phenotype of pks1, pks2 and PKS1OX. The fact that in the single mutants there are only small changes in the phyA′/phyA″ ratio, while in the double mutant the ratio is considerably affected, indicates that PKS1 or PKS2 act redundantly with each other in this regard.