Cylindrical containers agitated by orbital shaking are being developed as simple and cost-effective bioreactor systems for the cultivation of mammalian cells. Here the oxygen transfer capacities of containers with nominal volumes from 50 mL to 2000 L were determined, and the operating parameters influencing oxygen transfer were investigated. In general, the shaking speed necessary for efficient oxygen transfer diminished as the container size increased. At shaking speeds suitable for the growth of shear-sensitive cells, kLa values between 10 and 30 h−1 were typically achieved in small-scale (<1 L nominal volume) containers at shaking speeds above 120 rpm. A kLa value of 8 h−1 was measured at 75 rpm in a 200-L container with a working volume that was 50% of the nominal volume. In a 2000-L container with a working volume of 1000 L, a moderate kLa of 3 h−1 was obtained with a shaking speed of only 47 rpm. The free-surface area in 50-mL and 30-L containers was determined by photographic image analysis and computational fluid dynamic (CFD) simulation, respectively. The results showed that the increase in kLa at higher shaking speeds was mainly due to an increased kL value, highlighting the dominant effect of free-surface turbulence on gas transfer in orbitally shaken containers. The results demonstrated the feasibility of orbital shaking technology for the cultivation of mammalian cells at scales up to 1000 L.