The acknowledge takes place after every byte. The acknowledge bit allows the receiver to signal the transmitter that the byte was successfully received and another byte may be sent. All clock pulses, including the acknowledge ninth clock pulse, are generated by the host. The transmitter releases the SDA line during the acknowledge clock pulse so the receiver can pull the SDA line LOW and it remains stable LOW during the HIGH period of this clock pulse.

When SDA remains HIGH during the ninth clock pulse, this is the Not Acknowledge signal. The host can then generate either a STOP to abort the transfer or a repeated START to start a new transfer.