When using the I/O specific commands IN and OUT, the I/O addresses 0x00 - 0x3F must be used. When addressing I/O Registers as data space using LD and ST instructions, 0x20 must be added to these offset addresses.

In Transmit mode, TWDR contains the next byte to be transmitted. In Receive mode, the TWDR contains the last byte received. It is writable while the TWI is not in the process of shifting a byte. This occurs when the TWI Interrupt Flag (TWINT) is set by hardware. Note that the Data Register cannot be initialized by the user before the first interrupt occurs. The data in TWDR remains stable as long as TWINT is set. While data is shifted out, data on the bus is simultaneously shifted in. TWDR always contains the last byte present on the bus, except after a wake up from a sleep mode by the TWI interrupt. In this case, the contents of TWDR is undefined. In the case of a lost bus arbitration, no data is lost in the transition from Master to Slave. Handling of the ACK bit is controlled automatically by the TWI logic, the CPU cannot access the ACK bit directly.