The data on SDA line must be stable during the HIGH period of the clock signal (SCL). In other words, state of the data line can only be changed when clock signal is LOW. START and STOP conditions classify the beginning and the end of the data transfer session. A START condition is defined as the SDA signal transitioning from and the end of the data transfer session. A START condition is defined as the SDA signal transitioning from HIGH to LOW while SCL line is HIGH. A STOP condition is defined as the SDA transitioning from LOW to HIGH while SCL is HIGH. The bus leader always generates START and STOP conditions. The bus is considered to be busy after a START condition and free after a STOP condition. During data transmission, the bus leader can generate repeated START conditions. First START and repeated START conditions are functionally equivalent.

Each byte of data has to be followed by an acknowledge bit. The acknowledge related clock pulse is generated by the leader. The leader releases the SDA line (HIGH) during the acknowledge clock pulse. The device pulls down the SDA line during the 9th clock pulse, signifying an acknowledge. The device generates an acknowledge after each byte has been received.

There is one exception to the acknowledge after every byte rule. When the leader is the receiver, the receiver must indicate to the transmitter an end of data by not acknowledging (negative acknowledge) the last byte clocked out of the follower. This negative acknowledge still includes the acknowledge clock pulse (generated by the leader), but the SDA line is not pulled down.