Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (RBCS) is encoded by a nuclear RBCS multigene family in many plant species. The contribution of the RBCS multigenes to accumulation of Rubisco holoenzyme and photosynthetic characteristics remains unclear. T-DNA insertion mutants of RBCS1A (rbcs1a-1) and RBCS3B (rbcs3b-1) were isolated among the four Arabidopsis RBCS genes, and a double mutant (rbcs1a3b-1) was generated. RBCS1A mRNA was not detected in rbcs1a-1 and rbcs1a3b-1, while the RBCS3B mRNA level was suppressed to ∼20% of the wild-type level in rbcs3b-1 and rbcs1a3b-1 leaves. As a result, total RBCS mRNA levels declined to 52, 79, and 23% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. Rubisco contents showed declines similar to total RBCS mRNA levels, and the ratio of Rubisco-nitrogen to total nitrogen was 62, 78, and 40% of the wild-type level in rbcs1a-1, rbcs3b-1, and rbcs1a3b-1, respectively. The effects of RBCS1A and RBCS3B mutations in rbcs1a3b-1 were clearly additive. The rates of CO2 assimilation at ambient CO2 of 40 Pa were reduced with decreased Rubisco contents in the respective mutant leaves. Although the RBCS composition in the Rubisco holoenzyme changed, the CO2 assimilation rates per unit of Rubisco content were the same irrespective of the genotype. These results clearly indicate that RBCS1A and RBCS3B contribute to accumulation of Rubisco in Arabidopsis leaves and that these genes work additively to yield sufficient Rubisco for photosynthetic capacity. It is also suggested that the RBCS composition in the Rubisco holoenzyme does not affect photosynthesis under the present ambient [CO2] conditions.