The large, homologous proteins Bassoon (Bsn) and Piccolo are part of the cytomatrix at the active zone (AZ) of conventional chemical synapses, where they can partially compensate for each other’s function. At the highly specialized photoreceptor ribbon synapses, Piccolino (Pclo), a C-terminally truncated splice variant of Piccolo, is the functional partner of Bsn. In earlier studies, we detected free-floating photoreceptor synaptic ribbons and disrupted AZs in Bsn single knockout (sKO) mice1 and a loss of the plate‑like ribbon structure, but attached photoreceptor synaptic ribbons, in Pclo sKO mice2. In the present study, we generated a new conditional mouse line with a double knockout (dKO) of both Bsn and Pclo (Bsn/Pclo dKO) in rod photoreceptors to test for a potential cooperativity of the two proteins at the rod photoreceptor ribbon synapse. In Bsn/Pclo dKO, we observed a synaptic phenotype that closely resembles a combination of Bsn and Pclo sKOs: small spherical free-floating synaptic ribbons and disrupted AZs. In addition, we see phenotypes that were not present in sKO mice: sprouting and ectopic synapses of horizontal and bipolar cells, and most surprisingly, a decrease in retinal thickness, indicating a loss of rod photoreceptors. Rod photoreceptor degeneration as a result of Bsn or Pclo disruption has not been previously observed.1 Ryl M. et al. FASEB J. (2021) doi: 10.1096/fj.202001962R.2 Müller T. et al. J Neurosci. (2019) doi: 10.1523/JNEUROSCI.2038-18.2019.