The duration of access periods and the availability of virus in source plants are two factors that influence the transmission of barley yellow dwarf virus (BYDV) by its aphid vectors. This study was conducted to quantify the relationships among acquisition access period (AAP), virus titer in infected oats, and transmission of three isolates of BYDV from New York by two aphid vector species. Thirteen AAPs, ranging from 15 min to 72 hr, were examined, and virus titer was quantified from each virus source leaf by using enzyme-linked immunosorbent assay (ELISA). Two leaves from each plant were used as independent virus sources to test the effect of leaf age, in addition to virus titer, on acquisition efficiency. The older leaf on each source plant almost always contained less virus. The NY isolates of BYDV, RPV and PAV, were acquired by Rhopalosiphum padi within a 15-min AAP; however, a 1- to 2-hr or 2- to 3-hr AAP was required for 50% of the aphids to transmit PAV or RPV, respectively. The difference in virus titer among source leaves did not affect the ability of R. padi to transmit RPV, but did influence the transmission of PAV. Sitobion avenae required a 30-min AAP to acquire the MAV and PAV isolates of BYDV. Fifty percent of the aphids were able to transmit MAV or PAV after a 4- to 6-hr or 10- to 12-hr AAP, respectively. The ability of S. avenae to transmit MAV and PAV was significantly lower for older leaves. Analyses of the transmission and titer data revealed that the lower virus content of the older leaves accounted for the significant reduction in virus transmission by S. avenae. The transmission efficiency of various BYDV isolates is differentially influenced by several factors including aphid vector, length of acquisition feeding period, and physiological age of source tissue. In addition, our results suggest that virus titer, as it is affected by age and infection stage of the source tissue, can have a strong influence on acquisition and transmission efficiency of aphid vectors.