Dominant mutations in the gene encoding the ubiquitously-expressed splicing factor PRPF31 cause retinitis pigmentosa, a form of hereditary retinal degeneration, with reduced penetrance. We and others have previously shown that penetrance is tightly correlated with PRPF31 expression, as lymphoblastoid cell lines (LCLs) from affected patients produce less abundant PRPF31 transcripts than LCLs from their unaffected relatives carrying the same mutation. We have investigated the genetic elements determining the variable expression of PRPF31, and therefore possibly influencing the penetrance of its mutations, by quantifying PRPF31 mRNA levels in LCLs derived from 15 CEPH families (200 individuals), representative of the general population. We found that PRPF31 transcript abundance was a highly variable and highly heritable character. Moreover, by linkage analysis we showed that PRPF31 expression was significantly associated with at least one expression quantitative trait locus (eQTL), spanning a 8.2-Mb region on chromosome 14q21-23. We also investigated a previously mapped penetrance factor located near PRPF31 itself in LCLs from individuals belonging to selected families segregating PRPF31 mutations that displayed reduced penetrance. Our results indicate that, despite its constant association with the non-mutant allele, this factor was able to modulate the expression of both PRPF31 alleles. Furthermore, we showed that LCLs from affected patients have less PRPF31 RNA than those of asymptomatic patients, even at the pre-splicing stage. Altogether, these data demonstrate that PRPF31 mRNA expression and consequently the penetrance of PRPF31 mutations is managed by diffusible compounds encoded by at least two modifiers, acting in a co-regulatory system on both PRPF31 alleles during transcription.