Two identical sister copies of eukaryotic chromosomes are synthesized during S 
phase. To facilitate their recognition as pairs for segregation in mitosis, 
sister chromatids are held together from their synthesis onward by the 
chromosomal cohesin complex. Replication fork progression is thought to be 
coupled to establishment of sister chromatid cohesion, facilitating 
identification of replication products, but evidence for this has remained 
circumstantial. Here we show that three proteins required for sister chromatid 
cohesion, Eco1, Ctf4, and Ctf18, are found at, and Ctf4 travels along 
chromosomes with, replication forks. The ring-shaped cohesin complex is loaded 
onto chromosomes before S phase in an ATP hydrolysis-dependent reaction. 
Cohesion establishment during DNA replication follows without further cohesin 
recruitment and without need for cohesin to re-engage an ATP hydrolysis motif 
that is critical for its initial DNA binding. This provides evidence for 
cohesion establishment in the context of replication forks and imposes 
constraints on the mechanism involved.